CN111793136A

CN111793136A - NMP22 antibody pair and application thereof

Info

Publication number: CN111793136A
Application number: CN202010913875.6A
Authority: CN
Inventors: 刘继来; 程勇; 王征; 刘静
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

Abstract

The invention discloses an NMP22 antibody pair and application thereof, which comprises a monoclonal antibody AntiNMP22_ N combined with an N-terminal epitope of NMP22 and a monoclonal antibody AntiNMP22_ C combined with a C-terminal epitope of NMP22, specifically recognizes the AntiNMP22_ C by a chemiluminescence detection method, and is used for detecting the content of NMP 22. The invention uses the AntiNMP22_ N, AntiNMP22_ C antibody pair combined with the NMP22 epitope to detect the expression condition of NMP22 in a patient sample by using a chemiluminescence detection method to diagnose the bladder disease of the patient so as to indicate the risk of the patient suffering from bladder cancer.

Description

NMP22 antibody pair and application thereof

Technical Field

The invention relates to the field of urinary system disease diagnosis, in particular to an NMP22 antibody pair and application thereof.

Background

Among urinary system tumors, the incidence rate of bladder cancer is the leading position, and in recent years, the incidence rate of bladder cancer tends to increase. Most bladder cancer patients are diagnosed with well-differentiated or moderately differentiated non-muscle invasive bladder cancer, wherein about 10% of patients eventually develop muscle invasive bladder cancer or metastatic bladder cancer, and about 50% of tumors may recur within 2 years and about 10% -15% of recurrent tumors have an increasing tendency in malignancy among various surgical treatments for bladder preservation, so that the discovery of tumor recurrence in the early stage of long-term follow-up is one of the keys to successful bladder tumor treatment.

At present, specific, sensitive and stable early diagnosis markers are searched for, and play a positive role in early diagnosis, treatment and prognosis judgment of bladder cancer. Cystoscopy and biopsy are the diagnostic gold standard for bladder cancer, but are invasive tests, have high economic cost and are at risk of urinary tract infection.

It was found that the concentration of nuclear matrix protein (nuclear matrix protein) in the cells of patients with bladder cancer was 25 times higher than that of normal cells. NMP22 is released from cell nucleus after apoptosis, and exists in urine in the form of soluble complex or fragment, and the urinary NMP22 concentration level of bladder cancer patient is 20-80 times of that of non-cancer control group. NMP22 is a urine detectable bladder tumor marker studied abroad, and is described in detail in the literature "specific nuclear protein and its associated proteins with the polar region of the diagnostic reagents: distribution in a human/hamsterhybrid cell" and in the literature "Use of a new tu maker, url NM P22, in the detection of the clinical reporting of the clinical cancer by the Food and Drug Administration (FDA), and the detection of NMP22 tumor marker in bladder cancer plays a positive role in the diagnosis and prognosis of bladder cancer.

Disclosure of Invention

In view of the above-mentioned drawbacks or deficiencies in the prior art, it would be desirable to provide a pair of NMP22 antibodies and uses thereof.

According to the technical scheme provided by the embodiment of the application, the NMP22 antibody pair comprises a monoclonal antibody AntiNMP22_ N combined with an amino acid sequence shown as SEQ ID NO. 2 of an N-terminal epitope of NMP22 and a monoclonal antibody AntiNMP22_ C combined with an amino acid sequence shown as SEQ ID NO. 3 of a C-terminal epitope of NMP22, wherein the AntiNMP22_ N comprises a light chain variable region of an amino acid sequence shown as SEQ ID NO. 7 and a heavy chain variable region of an amino acid sequence shown as SEQ ID NO. 11, and the AntiNMP22_ C comprises a light chain variable region of an amino acid sequence shown as SEQ ID NO. 17 and a heavy chain variable region of an amino acid sequence shown as SEQ ID NO. 21.

In the invention, the light chain of the AntiNMP22_ N comprises three amino acid sequences of LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6, the heavy chain of the AntiNMP22_ N comprises three amino acid sequences of HCDR1 shown as SEQ ID NO. 8, HCDR2 shown as SEQ ID NO. 9 and HCDR3 shown as SEQ ID NO. 10,

the light chain of the AntiNMP22_ C comprises three amino acid sequences of LCDR1 shown as SEQ ID NO. 14, LCDR2 shown as SEQ ID NO. 15 and LCDR3 shown as SEQ ID NO. 16, and the heavy chain of the AntiNMP22_ C comprises three amino acid sequences of HCDR1 shown as SEQ ID NO. 18, HCDR2 shown as SEQ ID NO. 19 and HCDR3 shown as SEQ ID NO. 20.

In the present invention, the AntiNMP22_ N and the AntiNMP22_ C are IgG type monoclonal antibodies, and the AntiNMP22_ N and the AntiNMP22_ C can be converted to NMP22 binding fragments Fab, F (ab ') 2, Fab', scFv, di-scFv.

In the invention, the AntiNMP22_ N is a capture antibody, and the AntiNMP22_ C antibody is a detection antibody.

In the invention, the detection label of the AntiNMP22_ C is enzyme, fluorophore or radioisotope.

In the invention, the AntiNMP22_ N and the AntiNMP22_ C can be recombined with detection marker protein, and the detection marker protein is horseradish peroxidase, alkaline phosphatase, luciferase and beta-galactosidase.

In the present invention, the amount of NMP22 in the biological sample is detected by immunological detection methods including enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection, western blot detection and Immunohistochemical (IHC) detection.

A chemiluminescence detection kit comprises an AntiNMP22_ N combined with an N-terminal epitope of NMP22 and an AntiNMP22_ C antibody pair combined with a C-terminal epitope of MP22, specifically recognizes the AntiNMP22_ C by a chemiluminescence detection method, and is used for detecting the content of NMP 22.

To sum up, the beneficial effect of this application: the invention uses the AntiNMP22_ N, AntiNMP22_ C antibody pair combined with the NMP22 epitope to detect the expression condition of NMP22 in a patient sample by using a chemiluminescence detection method to diagnose the bladder disease of the patient so as to indicate the risk of the patient suffering from bladder cancer.

Drawings

Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:

FIG. 1 is a schematic diagram of the reaction principle of the paired antibodies of the present invention;

FIG. 2 is a diagram showing the construction of pcDNA3.1-NL-LFc vector of the present invention;

FIG. 3 is the construction of the pcDNA3.1-NH-HFc vector of the present invention;

FIG. 4 is a purification map of recombinant AntiNMP22_ N protein according to the present invention;

FIG. 5 shows the construction of pcDNA3.1-CL-LFc vector according to the invention;

FIG. 6 is a diagram showing the construction of the pcDNA3.1-CH-HFc vector of the present invention;

FIG. 7 is a purification map of recombinant AntiNMP22_ C protein according to the present invention;

FIG. 8 is a schematic diagram of the performance verification operation of the kit of the present invention;

FIG. 9 is a first measurement curve of dose-response curve validation in performance index assessment according to the present invention;

FIG. 10 is a second measurement curve of dose-response curve validation in performance index assessment according to the present invention;

FIG. 11 is a third measurement curve for dose-response curve validation in performance index assessment in accordance with the present invention;

FIG. 12 is a schematic diagram showing the correlation between the detection concentration results of the kit of the present invention and the market kit.

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 in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

An NMP22 antibody pair, comprising a monoclonal antibody AntiNMP22_ N combined with the amino acid sequence shown in SEQ ID NO. 2 as the N-terminal epitope of NMP22 and a monoclonal antibody AntiNMP22_ C combined with the amino acid sequence shown in SEQ ID NO. 3 as the C-terminal epitope of NMP22, wherein the AntiNMP22_ 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 AntiNMP22_ 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. The light chain of the AntiNMP22_ N comprises three amino acid sequences of LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5, LCDR3 as shown in SEQ ID NO. 6, the heavy chain of the AntiNMP22_ N comprises three amino acid sequences of HCDR1 as shown in SEQ ID NO. 8, HCDR2 as shown in SEQ ID NO. 9, and HCDR3 as shown in SEQ ID NO. 10, the light chain of the AntiNMP22_ C comprises three amino acid sequences of LCDR1 as shown in SEQ ID NO. 14, LCDR2 as shown in SEQ ID NO. 15, and LCDR3 as shown in SEQ ID NO. 16, the heavy chain of the AntiNMP22_ C comprises three amino acid sequences of HCDR1 as shown in SEQ ID NO. 18, HCDR2 as shown in SEQ ID NO. 19, and HCDR3 as shown in SEQ ID NO. 20. The AntiNMP22_ N and the AntiNMP22_ C are IgG type monoclonal antibodies, and the AntiNMP22_ N and the AntiNMP22_ C can be converted into NMP22 binding fragments Fab, F (ab ') 2, Fab', scFv and di-scFv. The AntiNMP22_ N is a capture antibody, and the AntiNMP22_ C antibody is a detection antibody. The detection label of the AntiNMP22_ C is enzyme, fluorescent group or radioactive isotope. The AntiNMP22_ N and the AntiNMP22_ C can be expressed by recombination with detection marker proteins, wherein the detection marker proteins are horseradish peroxidase, alkaline phosphatase, luciferase and beta-galactosidase. The amount of NMP22 in biological samples was detected by immunological detection methods including enzyme linked immunosorbent assay (ELISA), chemiluminescent detection, western blot detection and Immunohistochemical (IHC) detection.

Example 1

Preparation of monoclonal antibody AntiNMP22_ N and monoclonal antibody AntiNMP22_ C

a) The N-terminal epitope (SEQ ID NO:2) of NMP22 and the C-terminal epitope (SEQ ID NO:3) of NMP22 were synthesized separately, and mice immunized after coupling with KLH were 8-week-old female Balb/C mice, respectively.

b) The immunization method comprises the following steps: mice were immunized 4 times with 100ug of antigen, each time at 4 weeks intervals.

c) Cell fusion: mixing the immunized mouse spleen cells and mouse myeloma cells according to the ratio of 1:1, washing the mixture in a 50ml centrifuge tube for 1 time by using a serum-free incomplete culture solution, centrifuging the mixture at 1000rpm/min for 10 minutes, discarding supernatant, completely sucking residual liquid by using a pipette (in order to avoid influencing the concentration of PEG), and lightly flicking the bottom of the centrifuge tube to ensure that cell precipitation is slightly loosened.

d) 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.

e) 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.

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

g) 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.

h) Subpackaging 96-well cell culture plate (with feeder cell layer in the well plate) 0.1-0.15ml per well (or subpackaging 24-well plate with 1.0-1.5ml per well), and culturing at 37 deg.C in 6% CO2 incubator.

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

j) Changing out HAT culture medium after 7-10 days;

k) the growth of the hybridoma cells was frequently observed and the activity of the supernatant was measured by ELISA when the cells had grown to a well bottom area of 1/10 or more.

l) through multiple rounds of screening and limiting dilution, pure, potent clones are finally obtained.

Example 2

Screening of monoclonal antibody AntiNMP22_ N and monoclonal antibody AntiNMP22_ C

The cloned antibodies obtained in example 1 were screened for N-terminal antigen (SEQ ID NO:2) and C-terminal antigen (SEQ ID NO:3), respectively. Finally, antibody pairs AntiNMP22_ N and AntiNMP22_ C with optimal activity and pairing, respectively, were obtained, wherein AntiNMP22_ N was able to specifically bind to the N-terminal antigen of NMP22(SEQ ID NO:2) and not to the C-terminal antigen of NMP22(SEQ ID NO: 3). AntiNMP22_ C specifically binds to the C-terminal antigen of NMP22(SEQ ID NO:3) and not to the N-terminal antigen of NMP22(SEQ ID NO: 2).

NMP22 full antigen (SEQ ID NO:1) coating and chemiluminescence method verification

NMP22 was coated with the N-terminal antigen (SEQ ID NO:2) and detected by chemiluminescence.

NMP22 was coated with the C-terminal antigen (SEQ ID NO:3) and detected by chemiluminescence.

TABLE 1

	Whole antigen (SEQ ID NO:1)	N-terminal antigen (SEQ ID NO:2)	C-terminal antigen (SEQ ID NO:3)
				Blank group	6798	6953	6622
AntiNMP22_N 2ul	92302	83290	6991
				AntiNMP22_N 10ul	402993	387862	7099
AntiNMP22_N 50ul	1840516	1472154	7041
				AntiNMP22_C 10ul	89290	6832	86860
AntiNMP22_C 20ul	403333	6502	391525
				AntiNMP22_C 50ul	1823704	6898	1453347

As can be seen from table 1, it was confirmed by this experiment that the anti-nmp 22_ N in the antibody pair of the present invention recognizes the N-terminal antigen and does not recognize the C-terminal antigen. The AntiNMP22_ C in the antibody pair of the invention can recognize C-terminal antigen and can not recognize N-terminal antigen.

Example 3

Recombinant expression of N-terminal antibody AntiNMP22_ N of NMP22 and C-terminal antibody AntiNMP22_ C of NMP22

a. N-terminal antibody AntiNMP22_ N of recombinant expression NMP22

After sequencing the hybridomas of AntiNMP22_ N in the antibody pairs obtained in example 2, the light chain variable region sequence (SEQ ID NO:7) and the heavy chain variable region (SEQ ID NO:11) of antibody AntiNMP22_ N were obtained. After optimizing the sequence, the light chain variable region was cloned into pcDNA3.1-LFc vector to obtain pcDNA3.1-NL-LFc (FIG. 2). The variable region of the heavy chain was cloned into pcDNA3.1-HFc vector to obtain pcDNA3.1-NH-HFc (FIG. 3). pcDNA3.1-LFc and pcDNA3.1-HFc already contain human light and heavy chain constant regions. Wherein the nucleic acid sequence of NL (light chain variable region) is SEQ ID NO:12 and the nucleic acid sequence of NH (heavy chain variable region) is SEQ ID NO: 13. pcDNA3.1-NL-LFc and pcDNA3.1-NH-HFc were co-transfected into CHO-S cells at 1:1 to recombinantly express the N-terminal antibody AntiNMP22_ N against NMP22 (FIG. 4).

Note:

lane M in FIG. 2 is DNA Marker;

Lane 1：pcDNA3.1-NL-LFc Plasmid；

Lane 2:NL

lane M in FIG. 3 is DNA Marker;

Lane 1：pcDNA3.1--HFc Plasmid；

Lane 2:NH

lane M in fig. 4: marker;

Lane 1：AntiNMP22_N

b. n-terminal antibody AntiNMP22_ C of recombinant expression NMP22

After sequencing the hybridomas of AntiNMP22_ C in the antibody pairs obtained in example 2, the light chain variable region sequence (SEQ ID NO:17) and the heavy chain variable region (SEQ ID NO:21) of antibody AntiNMP22_ C were obtained. After the sequence was optimized, the light chain variable region was cloned into pcDNA3.1-LFc vector to obtain pcDNA3.1-CL-LFc (FIG. 5). The variable region of the heavy chain was cloned into pcDNA3.1-HFc vector to obtain pcDNA3.1-CH-HFc (FIG. 6). pcDNA3.1-LFc and pcDNA3.1-HFc already contain human light and heavy chain constant regions. Wherein the nucleic acid sequence of CL (light chain variable region) is SEQ ID NO:22, and the nucleic acid sequence of NH (heavy chain variable region) is SEQ ID NO: 23. pcDNA3.1-CL-LFc and pcDNA3.1-CH-HFc were co-transfected into CHO-S cells at 1:1 to recombinantly express the C-terminal antibody AntiNMP22_ C against NMP22 (FIG. 7).

Note:

lane M in FIG. 5 is DNA Marker;

Lane 1：pcDNA3.1-CL-LFc Plasmid；

Lane 2:CL

lane M in FIG. 6 is DNA Marker;

Lane 1：pcDNA3.1-CH-HFc Plasmid；

Lane 2:CH

lane M in fig. 7: marker;

Lane 1：AntiNMP22_C

example 4

Preparation and verification of antibody pair participating chemiluminescence kit

According to the existing production process of a chemiluminescence method diagnostic reagent, the AntiNMP22_ C is used as a capture antibody to be coated to form an NMP22 coated plate, the AntiNMP22_ N label is used as a binding antibody to be coated to form an NMP22 enzyme conjugate, and the NMP22 antigen is prepared into a series of calibrators (0U/ml, 5U/ml, 10U/ml, 25U/ml, 50U/ml and 100U/ml) to form a complete kit and carry out a series of verifications.

Referring to the industrial standard of the YY/T1175-2010 tumor marker quantitative determination reagent (kit) chemiluminescence method and the technical requirements of the existing NMP22 kit manufacturers in the market, the performance verification index of the NMP22 wash-free detection reagent is formulated

1. Performance index

1.1 minimum detection Limit

Should not be higher than 4U/ml.

1.2 dose-response Curve Linearity

In the linear interval of 0U/ml-100U/ml, the linear correlation coefficient (r) of the dose-response curve should be not less than 0.9900.

1.3 accuracy

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

1.4 precision

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

2. Inspection method

2.1 minimum detection Limit

The relative luminous intensity of a 20-hole zero calibrator (0U/ml) is measured in parallel, the average value x and the standard deviation SD of the luminous value are calculated, the concentration value corresponding to the luminous value x +2 xSD is the lowest detection limit when the dose-response curve is used for calculating, and the result meets the requirement of 1.1.

2.2 dose-response Curve linearity

The reference substances (S0-S5, the concentration is 0U/ml, 5U/ml, 10U/ml, 25U/ml, 50U/ml and 100U/ml in sequence) of the multi-well determination kit are fitted by four parameters, and the correlation coefficient (r) of a dose-response curve meets the requirement of 1.2.

2.3 accuracy

Preparing NMP22 antigen into a standard solution (80U/ml) according to a volume ratio of 1: 9 is added into a low-value sample (2-5U/ml) with known concentration, detection is carried out on the kit, the recovery rate R is calculated according to the formula (1), and the result meets the regulation of 1.3.

In the formula: r-recovery rate;

v-volume of standard solution added;

v0 — volume of low value sample;

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

c0-concentration of low sample;

cs-concentration of standard solution.

2.4 precision

The CV was calculated by measuring 10 wells of each of the quality control Q1 and the quality control Q2 in parallel according to the formula, and the result was determined to meet the 1.4 rule.

In the formula:

average value of concentration values determined by quality control

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

3. Experimental configuration

3.1 NMP22 coated plate configuration

AntiNMP22_ C was added as a capture antibody to the coating buffer (0.05mol PBS) at a ratio of 1 ug/ml. Adding 100ul of the blank luminescent plate holes, 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 liquid in the plate, and drying to prepare an NMP22 coated plate.

3.2 NMP22 enzyme conjugate configuration

AntiNMP22_ N was added as a conjugated antibody to an enzyme diluent (50Mmol Tris + 1% BSA) at a ratio of 1/2000.

3.3 calibrator configuration

NMP22 antigen (Jiufonguda, 20191221, 3KU/ml) was diluted 6 gradients (0U/ml, 5U/ml, 10U/ml, 25U/ml, 50U/ml, 100U/ml) with antigen diluent (0.5mol PBS + 1% BSA)

3.4 recovery Standard solution preparation

Diluting NMP22 antigen (Jiufenguda, 20191221, 3KU/ml) to 80U/ml with antigen diluent (0.5mol PBS + 1% BSA) as standard solution, selecting 2-5U/ml normal human serum as low value sample

3.5 quality control product configuration

NMP22 antigen (Jiufonguda, 20191221, 3KU/ml) was diluted with antigen diluent (0.5mol PBS + 1% BSA) to QC1 (20U/ml. + -. 10%), QC2 (65U/ml. + -. 10%)

4. Sample application operation

(1) Respectively taking the 3.3-3.5NMP22 calibrator, recovery rate specimen, quality control material and specific sample each 100 μ l, adding into the corresponding coated plate hole of 3.1, and shaking with oscillator for 30 s to mix them thoroughly

(2) Cover with a sealing plate membrane, incubate at 37 ℃ for 30 minutes

(3) Removed and the plate washed 3 times with application wash (0.5mol PBS + 0.025% T-20)

(4) 100. mu.L of the 3.2 NMP22 enzyme conjugate prepared as described above was added to each well and the mixture was shaken for 30 seconds with a shaker

(5) Cover with a sealing plate membrane, incubate at 37 ℃ for 30 minutes

(6) Removed and the plate washed 3 times with application wash (0.5mol PBS + 0.025% T-20)

(7) 100ul of substrate solution (purchased from ThermoFisher T2142) was added to each well

(8) Chemiluminescence intensity (RLU) was measured with a Zhongshengbuck BHP9504 chemiluminescence apparatus

5. Performance index verification result

5.1 minimum detection Limit

TABLE 2

As can be seen from Table 2, the minimum detection limit is 1.392U/ml, which is not higher than 4U/ml, and meets the index.

5.2 dose-response Curve Linearity

TABLE 3

	First measurement	Second measurement	The third measurement
				Linear correlation coefficient R	1.0000	0.9996	0.9998

As can be seen from Table 3, FIG. 9, FIG. 10 and FIG. 11, the linear correlation coefficient r of the NMP22 antigen curve in the concentration range of 0-100U/ml measured three times in succession was greater than 0.9990, and the index was satisfied.

5.3 accuracy

TABLE 4

CS(pmol/L)	C0(pmol/L)	Sample C luminescence value	C(pmol/L)	Recovery (%)
					80	3.3	220010	11.3	104.13

As can be seen from Table 4, the recovery rate of the kit detection is in the range of 0.85-1.15, which meets the index.

5.4 precision

TABLE 5

As can be seen from Table 5, the results of three consecutive parallel determinations of QC1 QC2 are not more than 10% and meet the index.

From the above, the performance indexes of the NMP22 kit, such as dose-response curve, accuracy, uniformity and the like, all meet the requirements of the prior art

Example 5

Comparison test of the antibody kit of the invention and the existing kits in the market

Randomly selecting 40 human specimens, preparing a kit by using the antibody of the invention and detecting the specimens by using the conventional NMP22 kit in the market, and comparing the correlation

The results of the comparison of 40 human specimens are as follows:

TABLE 6

	The invention	Contrast agent		The invention	Contrast agent		The invention	Contrast agent		The invention	Contrast agent
													1	8.85	8.07	11	7.22	7.40	21	50.70	51.99	31	5.03	5.16
2	2.42	2.56	12	2.49	2.63	22	6.95	6.34	32	3.30	3.49
												3	6.01	5.98	13	9.90	9.03	23	1.68	1.67	33	0.81	0.80
4	40.09	36.58	14	83.85	83.39	24	5.30	5.60	34	3.80	3.89
												5	2.61	2.59	15	7.31	7.72	25	2.73	2.49	35	6.10	5.56
6	3.33	3.42	16	5.61	5.75	26	2.80	2.88	36	1.91	1.96
												7	7.54	6.88	17	1.61	1.47	27	10.83	10.79	37	9.87	9.83
8	43.55	43.31	18	6.93	7.32	28	6.58	6.95	38	1.91	1.90
												9	10.31	10.57	19	9.59	9.54	29	2.01	2.07	39	7.35	6.70
10	6.65	6.62	20	1.35	1.42	30	9.86	10.12	40	8.87	9.37

As shown in Table 6 and FIG. 9, the correlation R between the results of the concentrations measured by the present invention and the results of the concentrations measured by the existing kits on the market²Is 0.9983.

As can be seen from the above, the detection result of the clinical sample prepared based on the reagent of the invention is consistent with the detection result of the existing chemiluminescence detection reagent in the market.

Example 6

The sequence of NMP22 is shown in SEQ ID NO 1. Because NMP22 has a large antigen, the difficulty of preparing the antigen is large. Meanwhile, no paired antibody for clinical detection of NMP22 has been reported so far. The NMP22 antigen was selected for the inventive split into two sequences, N-terminal (SEQ ID NO:2) and C-terminal (SEQ ID NO: 3). And the mice are immunized by the two sequences respectively, and antibody pairs with high specificity and high sensitivity are screened for AntiNMP22_ N and AntiNMP22_ C.

In a first embodiment, the present invention provides the core sequence of an NMP22 antibody pair (variable region of the antibody to the heavy and light chains). Wherein SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 in the light chain sequence and SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 in the heavy chain sequence of AntiNMP22_ N are the key positions for binding of NMP22 antigen N-terminal epitope (SEQ ID NO. 2). 14, 15, 16 in the light chain sequence and 18, 19, 20 in the heavy chain sequence of AntiNMP22_ C are the key positions for binding of NMP22 antigen C-terminal epitope (SEQ ID NO: 3).

The antibodies in the antibody pairs of the invention may be Fab fragments; may be a F (ab') 2 fragment; may be a Fab' fragment; may be a scFv fragment; may be a di-scFv fragment.

The antibodies in the antibody pairs of the invention are monoclonal antibodies.

The antibodies in the antibody pairs of the invention are IgG-type antibodies.

The antibody pair of the invention is a murine antibody or an antibody of murine origin.

The antibody AntiNMP22_ N in the antibody pair of the invention is a capture antibody and AntiNMP22_ C is a labeled antibody, which may be further coupled to a detectable label.

The antibody of the pair of antibodies of the invention, AntiNMP22_ C, is detectably labeled with a semienzyme or enzyme or a fluorophore or radioisotope.

The antibodies of the invention are used to detect the presence of NMP22(SEQ ID NO:1) in a sample, which according to one embodiment is a biological sample, preferably urine.

The antibody pair of the present invention was tested for the presence of NMP22(SEQ ID NO:1) by a sandwich immunological method. The present implementation class selects chemiluminescence methodology for detection. Therefore, the content of NMP22 in the biological sample can be accurately detected.

The antibody of the invention detects the existence and expression of NMP22 by a chemical light-emitting method for AntiNMP22_ N and AntiNMP22_ C.

The samples used in the in vitro methods of the invention are derived from subjects suffering from or at risk of: cystitis, bladder cancer.

The invention relates to application of the antibody of the invention to AntiNMP22_ N and AntiNMP22_ C in detecting NMP22 expression in a sample.

The present invention provides expression vectors pcdna3.1-AntiNMP22_ NL and pcdna3.1-AntiNMP22_ NH expressing AntiNMP22 comprising heavy and light chain polynucleotides encoding the antibody AntiNMP22_ N of the present invention.

The present invention provides expression vectors pcdna3.1-AntiNMP22_ CL and pcdna3.1-AntiNMP22_ CH expressing AntiNMP22 comprising heavy and light chain polynucleotides encoding the antibody AntiNMP22_ C of the present invention.

The invention discloses an expression vector for producing the antibody pair AntiNMP22_ N and AntiNMP22_ C.

The present invention provides at least one host cell comprising at least 2 expression vectors according to the invention.

The invention provides at least one host cell according to the invention for the preparation of an antibody pair of the invention.

The present invention provides a method for diagnosing a bladder disease, comprising the steps of: the above disclosed antibodies of the invention against AntiNMP22_ N (as capture antibody) and AntiNMP22_ C (as binding antibody) were used to detect NMP22 expression in patient samples using a chemiluminescent detection method. If the content of NMP22 is more than 10U/ml, the risk of bladder cancer is indicated.

The foregoing description is only exemplary of the preferred embodiments of the application and is provided for the purpose of illustrating the general principles of the technology and the like. Meanwhile, the scope of the invention according to the present application is not limited to the technical solutions in which the above-described technical features are combined in a specific manner, and also covers other technical solutions in which the above-described technical features or their equivalent are combined arbitrarily without departing from the inventive concept described above. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.

Sequence listing

<110> corridor sky light biotechnology Limited

<120> NMP22 antibody pair and application thereof

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<170>SIPOSequenceListing 1.0

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<211>2115

<212>PRT

<213> mouse (Mus musculus)

<400>1

Met Thr Leu His Ala Thr Arg Gly Ala Ala Leu Leu Ser Trp Val Asn

1 5 10 15

Ser Leu His Val Ala Asp Pro Val Glu Ala Val Leu Gln Leu Gln Asp

20 25 30

Cys Ser Ile Phe Ile Lys Ile Ile Asp Arg Ile His Gly Thr Glu Glu

35 40 45

Gly Gln Gln Ile Leu Lys Gln Pro Val Ser Glu Arg Leu Asp Phe Val

50 55 60

Cys Ser Phe Leu Gln Lys Asn Arg Lys His Pro Ser Ser Pro Glu Cys

65 70 75 80

Leu Val Ser Ala Gln Lys Val Leu Glu Gly Ser Glu Leu Glu Leu Ala

85 90 95

Lys Met Thr Met Leu Leu Leu Tyr His Ser Thr Met Ser Ser Lys Ser

100 105 110

Pro Arg Asp Trp Glu Gln Phe Glu Tyr Lys Ile Gln Ala Glu Leu Ala

115 120 125

Val Ile Leu Lys Phe Val Leu Asp His Glu Asp Gly Leu Asn Leu Asn

130 135 140

Glu Asp Leu Glu Asn Phe Leu Gln Lys Ala Pro Val Pro Ser Thr Cys

145 150 155 160

Ser Ser Thr Phe Pro Glu Glu Leu Ser Pro Pro Ser His Gln Ala Lys

165 170 175

Arg Glu Ile Arg Phe Leu Glu Leu Gln Lys Val Ala Ser Ser Ser Ser

180 185 190

Gly Asn Asn Phe Leu Ser Gly Ser Pro Ala Ser Pro Met Gly Asp Ile

195 200 205

Leu Gln Thr Pro Gln Phe Gln Met Arg Arg Leu Lys Lys Gln Leu Ala

210 215 220

Asp Glu Arg Ser Asn Arg Asp Glu Leu Glu Leu Glu Leu Ala Glu Asn

225 230 235 240

Arg Lys Leu Leu Thr Glu Lys Asp Ala Gln Ile Ala Met Met Gln Gln

245 250 255

Arg Ile Asp Arg Leu Ala Leu Leu Asn Glu Lys Gln Ala Ala Ser Pro

260 265 270

Leu Glu Pro Lys Glu Leu Glu Glu Leu Arg Asp Lys Asn Glu Ser Leu

275 280 285

Thr Met Arg Leu His Glu Thr Leu Lys Gln Cys Gln Asp Leu Lys Thr

290 295 300

Glu Lys Ser Gln Met Asp Arg Lys Ile Asn Gln Leu Ser Glu Glu Asn

305 310 315 320

Gly Asp Leu Ser Phe Lys Leu Arg Glu Phe Ala Ser His Leu Gln Gln

325 330 335

Leu Gln Asp Ala Leu Asn Glu Leu Thr Glu Glu His Ser Lys Ala Thr

340 345 350

Gln Glu Trp Leu Glu Lys Gln Ala Gln Leu Glu Lys Glu Leu Ser Ala

355 360 365

Ala Leu Gln Asp Lys Lys Cys Leu Glu Glu Lys Asn Glu Ile Leu Gln

370 375 380

Gly Lys Leu Ser Gln Leu Glu Glu His Leu Ser Gln Leu Gln Asp Asn

385 390 395 400

Pro Pro Gln Glu Lys Gly Glu Val Leu Gly Asp Val Leu Gln Leu Glu

405 410 415

Thr Leu Lys Gln Glu Ala Ala Thr Leu Ala Ala Asn Asn Thr Gln Leu

420 425 430

Gln Ala Arg Val Glu Met Leu Glu Thr Glu Arg Gly Gln Gln Glu Ala

435 440 445

Lys Leu Leu Ala Glu Arg Gly His Phe Glu Glu Glu Lys Gln Gln Leu

450 455 460

Ser Ser Leu Ile Thr Asp Leu Gln Ser Ser Ile Ser Asn Leu Ser Gln

465 470 475 480

Ala Lys Glu Glu Leu Glu Gln Ala Ser Gln Ala His Gly Ala Arg Leu

485 490 495

Thr Ala Gln Val Ala Ser Leu Thr Ser Glu Leu Thr Thr Leu Asn Ala

500 505 510

Thr Ile Gln Gln Gln Asp Gln Glu Leu Ala Gly Leu Lys Gln Gln Ala

515 520 525

Lys Glu Lys Gln Ala Gln Leu Ala Gln Thr Leu Gln Gln Gln Glu Gln

530 535 540

Ala Ser Gln Gly Leu Arg His Gln Val Glu Gln Leu Ser Ser Ser Leu

545 550 555 560

Lys Gln Lys Glu Gln Gln Leu Lys Glu Val Ala Glu Lys Gln Glu Ala

565 570 575

Thr Arg Gln Asp His Ala Gln Gln Leu Ala Thr Ala Ala Glu Glu Arg

580 585 590

Glu Ala Ser Leu Arg Glu Arg Asp Ala Ala Leu Lys Gln Leu Glu Ala

595 600 605

Leu Glu Lys Glu Lys Ala Ala Lys Leu Glu Ile Leu Gln Gln Gln Leu

610 615 620

Gln Val Ala Asn Glu Ala Arg Asp Ser Ala Gln Thr Ser Val Thr Gln

625 630 635 640

Ala Gln Arg Glu Lys Ala Glu Leu Ser Arg Lys Val Glu Glu Leu Gln

645 650 655

Ala Cys Val Glu Thr Ala Arg Gln Glu Gln His Glu Ala Gln Ala Gln

660 665 670

Val Ala Glu Leu Glu Leu Gln Leu Arg Ser Glu Gln Gln Lys Ala Thr

675 680 685

Glu Lys Glu Arg Val Ala Gln Glu Lys Asp Gln Leu Gln Glu Gln Leu

690 695 700

Gln Ala Leu Lys Glu Ser Leu Lys Val Thr Lys Gly Ser Leu Glu Glu

705 710 715 720

Glu Lys Arg Arg Ala Ala Asp Ala Leu Glu Glu Gln Gln Arg Cys Ile

725 730 735

Ser Glu Leu Lys Ala Glu Thr Arg Ser Leu Val Glu Gln His Lys Arg

740 745 750

Glu Arg Lys Glu Leu Glu Glu Glu Arg Ala Gly Arg Lys Gly Leu Glu

755 760 765

Ala Arg Leu Gln Gln Leu Gly Glu Ala His Gln Ala Glu Thr Glu Val

770 775 780

Leu Arg Arg Glu Leu Ala Glu Ala Met Ala Ala Gln His Thr Ala Glu

785 790 795 800

Ser Glu Cys Glu Gln Leu Val Lys Glu Val Ala Ala Trp Arg Glu Arg

805 810 815

Tyr Glu Asp Ser Gln Gln Glu Glu Ala Gln Tyr Gly Ala Met Phe Gln

820 825 830

Glu Gln Leu Met Thr Leu Lys Glu Glu Cys Glu Lys Ala Arg Gln Glu

835 840 845

Leu Gln Glu Ala Lys Glu Lys Val Ala Gly Ile Glu Ser His Ser Glu

850 855 860

Leu Gln Ile Ser Arg Gln Gln Asn Glu Leu Ala Glu Leu His Ala Asn

865 870 875 880

Leu Ala Arg Ala Leu Gln Gln Val Gln Glu Lys Glu Val Arg Ala Gln

885 890 895

Lys Leu Ala Asp Asp Leu Ser Thr Leu Gln Glu Lys Met Ala Ala Thr

900 905 910

Ser Lys Glu Val Ala Arg Leu Glu Thr Leu Val Arg Lys Ala Gly Glu

915 920 925

Gln Gln Glu Thr Ala Ser Arg Glu Leu Val Lys Glu Pro Ala Arg Ala

930 935 940

Gly Asp Arg Gln Pro Glu Trp Leu Glu Glu Gln Gln Gly Arg Gln Phe

945 950 955 960

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

965 970 975

Met Gly Asn Glu Leu Glu Arg Leu Arg Ala Ala Leu Met Glu Ser Gln

980 985 990

Gly Gln Gln Gln Glu Glu Arg Gly Gln Gln Glu Arg Glu Val Ala Arg

995 1000 1005

Leu Thr Gln Glu Arg Gly Arg Ala Gln Ala Asp Leu Ala Leu Glu Lys

1010 1015 1020

Ala Ala Arg Ala Glu Leu Glu Met Arg Leu Gln Asn Ala Leu Asn Glu

1025 1030 1035 1040

Gln Arg Val Glu Phe Ala Thr Leu Gln Glu Ala Leu Ala His Ala Leu

1045 1050 1055

Thr Glu Lys Glu Gly Lys Asp Gln Glu Leu Ala Lys Leu Arg Gly Leu

1060 1065 1070

Glu Ala Ala Gln Ile Lys Glu Leu Glu Glu Leu Arg Gln Thr Val Lys

1075 1080 1085

Gln Leu Lys Glu Gln Leu Ala Lys Lys Glu Lys Glu His Ala Ser Gly

1090 1095 1100

Ser Gly Ala Gln Ser Glu Ala Ala Gly Arg Thr Glu Pro Thr Gly Pro

1105 1110 1115 1120

Lys Leu Glu Ala Leu Arg Ala Glu Val Ser Lys Leu Glu Gln Gln Cys

1125 1130 1135

Gln Lys Gln Gln Glu Gln Ala Asp Ser Leu Glu Arg Ser Leu Glu Ala

1140 1145 1150

Glu Arg Ala Ser Arg Ala Glu Arg Asp Ser Ala Leu Glu Thr Leu Gln

1155 1160 1165

Gly Gln Leu Glu Glu Lys Ala Gln Glu Leu Gly His Ser Gln Ser Ala

1170 1175 1180

Leu Ala Ser Ala Gln Arg Glu Leu Ala Ala Phe Arg Thr Lys Val Gln

1185 1190 1195 1200

Asp His Ser Lys Ala Glu Asp Glu Trp Lys Ala Gln Val Ala Arg Gly

1205 1210 1215

Arg Gln Glu Ala Glu Arg Lys Asn Ser Leu Ile Ser Ser Leu Glu Glu

1220 1225 1230

Glu Val Ser Ile Leu Asn Arg Gln Val Leu Glu Lys Glu Gly Glu Ser

1235 1240 1245

Lys Glu Leu Lys Arg Leu Val Met Ala Glu Ser Glu Lys Ser Gln Lys

1250 1255 1260

Leu Glu Glu Arg Leu Arg Leu Leu Gln Ala Glu Thr Ala Ser Asn Ser

1265 1270 1275 1280

Ala Arg Ala Ala Glu Arg Ser Ser Ala Leu Arg Glu Glu Val Gln Ser

1285 1290 1295

Leu Arg Glu Glu Ala Glu Lys Gln Arg Val Ala Ser Glu Asn Leu Arg

1300 1305 1310

Gln Glu Leu Thr Ser Gln Ala Glu Arg Ala Glu Glu Leu Gly Gln Glu

1315 1320 1325

Leu Lys Ala Trp Gln Glu Lys Phe Phe Gln Lys Glu Gln Ala Leu Ser

1330 1335 1340

Thr Leu Gln Leu Glu His Thr Ser Thr Gln Ala Leu Val Ser Glu Leu

1345 1350 1355 1360

Leu Pro Ala Lys His Leu Cys Gln Gln Leu Gln Ala Glu Gln Ala Ala

1365 1370 1375

Ala Glu Lys Arg His Arg Glu Glu Leu Glu Gln Ser Lys Gln Ala Ala

1380 1385 1390

Gly Gly Leu Arg Ala Glu Leu Leu Arg Ala Gln Arg Glu Leu Gly Glu

1395 1400 1405

Leu Ile Pro Leu Arg Gln Lys Val Ala Glu Gln Glu Arg Thr Ala Gln

1410 1415 1420

Gln Leu Arg Ala Glu Lys Ala Ser Tyr Ala Glu Gln Leu Ser Met Leu

1425 1430 1435 1440

Lys Lys Ala His Gly Leu Leu Ala Glu Glu Asn Arg Gly Leu Gly Glu

1445 1450 1455

Arg Ala Asn Leu Gly Arg Gln Phe Leu Glu Val Glu Leu Asp Gln Ala

1460 1465 1470

Arg Glu Lys Tyr Val Gln Glu Leu Ala Ala Val Arg Ala Asp Ala Glu

1475 1480 1485

Thr Arg Leu Ala Glu Val Gln Arg Glu Ala Gln Ser Thr Ala Arg Glu

1490 1495 1500

Leu Glu Val Met Thr Ala Lys Tyr Glu Gly Ala Lys Val Lys Val Leu

1505 1510 1515 1520

Glu Glu Arg Gln Arg Phe Gln Glu Glu Arg Gln Lys Leu Thr Ala Gln

1525 1530 1535

Val Glu Gln Leu Glu Val Phe Gln Arg Glu Gln Thr Lys Gln Val Glu

1540 1545 1550

Glu Leu Ser Lys Lys Leu Ala Asp Ser Asp Gln Ala Ser Lys Val Gln

1555 1560 1565

Gln Gln Lys Leu Lys Ala Val Gln Ala Gln Gly Gly Glu Ser Gln Gln

1570 1575 1580

Glu Ala Gln Arg Leu Gln Ala Gln Leu Asn Glu Leu Gln Ala Gln Leu

1585 1590 1595 1600

Ser Gln Lys Glu Gln Ala Ala Glu His Tyr Lys Leu Gln Met Glu Lys

1605 1610 1615

Ala Lys Thr His Tyr Asp Ala Lys Lys Gln Gln Asn Gln Glu Leu Gln

1620 1625 1630

Glu Gln Leu Arg Ser Leu Glu Gln Leu Gln Lys Glu Asn Lys Glu Leu

1635 1640 1645

Arg Ala Glu Ala Glu Arg Leu Gly His Glu Leu Gln Gln Ala Gly Leu

1650 1655 1660

Lys Thr Lys Glu Ala Glu Gln Thr Cys Arg His Leu Thr Ala Gln Val

1665 1670 1675 1680

Arg Ser Leu Glu Ala Gln Val Ala His Ala Asp Gln Gln Leu Arg Asp

1685 1690 1695

Leu Gly Lys Phe Gln Val Ala Thr Asp Ala Leu Lys Ser Arg Glu Pro

1700 1705 1710

Gln Ala Lys Pro Gln Leu Asp Leu Ser Ile Asp Ser Leu Asp Leu Ser

1715 1720 1725

Cys Glu Glu Gly Thr Pro Leu Ser Ile Thr Ser Lys Leu Pro Arg Thr

1730 1735 1740

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

1745 1750 1755 1760

Gln Arg Leu Pro Pro Lys Val Glu Ser Leu Glu Ser Leu Tyr Phe Thr

1765 1770 1775

Pro Ile Pro Ala Arg Ser Gln Ala Pro Leu Glu Ser Ser Leu Asp Ser

1780 1785 1790

Leu Gly Asp Val Phe Leu Asp Ser Gly Arg Lys Thr Arg Ser Ala Arg

1795 1800 1805

Arg Arg Thr Thr Gln Ile Ile Asn Ile Thr Met Thr Lys Lys Leu Asp

1810 1815 1820

Val Glu Glu Pro Asp Ser Ala Asn Ser Ser Phe Tyr Ser Thr Arg Ser

1825 1830 1835 1840

Ala Pro Ala Ser Gln Ala Ser Leu Arg Ala Thr Ser Ser Thr Gln Ser

1845 1850 1855

Leu Ala Arg Leu Gly Ser Pro Asp Tyr Gly Asn Ser Ala Leu Leu Ser

1860 1865 1870

Leu Pro Gly Tyr Arg Pro Thr Thr Arg Ser Ser Ala Arg Arg Ser Gln

1875 1880 1885

Ala Gly Val Ser Ser Gly Ala Pro Pro Gly Arg Asn Ser Phe Tyr Met

1890 1895 1900

Gly Thr Cys Gln Asp Glu Pro Glu Gln Leu Asp Asp Trp Asn Arg Ile

1905 1910 1915 1920

Ala Glu Leu Gln Gln Arg Asn Arg Val Cys Pro Pro His Leu Lys Thr

1925 1930 1935

Cys Tyr Pro Leu Glu Ser Arg Pro Ser Leu Ser Leu Gly Thr Ile Thr

1940 1945 1950

Asp Glu Glu Met Lys Thr Gly Asp Pro Gln Glu Thr Leu Arg Arg Ala

1955 1960 1965

Ser Met Gln Pro Ile Gln Ile Ala Glu Gly Thr Gly Ile Thr Thr Arg

1970 1975 1980

Gln Gln Arg Lys Arg Val Ser Leu Glu Pro His Gln Gly Pro Gly Thr

1985 1990 1995 2000

Pro Glu Ser Lys Lys Ala Thr Ser Cys Phe Pro Arg Pro Met Thr Pro

2005 2010 2015

Arg Asp Arg His Glu Gly Arg Lys Gln Ser Thr Thr Glu Ala Gln Lys

2020 2025 2030

Lys Ala Ala Pro Ala Ser Thr Lys Gln Ala Asp Arg Arg Gln Ser Met

2035 2040 2045

Ala Phe Ser Ile Leu Asn Thr Pro Lys Lys Leu Gly Asn Ser Leu Leu

2050 2055 2060

Arg Arg Gly Ala Ser Lys Lys Ala Leu Ser Lys Ala Ser Pro Asn Thr

2065 2070 2075 2080

Arg Ser Gly Thr Arg Arg Ser Pro Arg Ile Ala Thr Thr Thr Ala Ser

2085 2090 2095

Ala Ala Thr Ala Ala Ala Ile Gly Ala Thr Pro Arg Ala Lys Gly Lys

2100 2105 2110

Ala Lys His

2115

<210>2

<211>26

<212>PRT

<213> mouse (Mus musculus)

<400>2

Pro Val Ser Glu Arg Leu Asp Phe Val Cys Ser Phe Leu Gln Lys Asn

1 5 10 15

Arg Lys His Pro Ser Ser Pro Glu Cys Leu

20 25

<210>3

<211>37

<212>PRT

<213> mouse (Mus musculus)

<400>3

Thr Pro Lys Lys Leu Gly Asn Ser Leu Leu Arg Arg Gly Ala Ser Lys

1 5 10 15

Lys Ala Leu Ser Lys Ala Ser Pro Asn Thr Arg Ser Gly Thr Arg Arg

20 25 30

SerPro Arg Ile Ala

35

<210>4

<211>12

<212>PRT

<213> mouse (Mus musculus)

<400>4

Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr

1 5 10

<210>5

<211>3

<212>PRT

<213> mouse (Mus musculus)

<400>5

Phe Ala Ser

1

<210>6

<211>9

<212>PRT

<213> mouse (Mus musculus)

<400>6

Gln Gln Gln Tyr Asn Thr Pro Leu Thr

1 5

<210>7

<211>113

<212>PRT

<213> mouse (Mus musculus)

<400>7

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

1 5 10 15

Gln Lys Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

Gln Tyr Asn Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210>8

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>8

Gly Tyr Thr Phe Thr Asn Ser Gly

1 5

<210>9

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>9

Ile Asn Thr Tyr Thr Gly Glu Pro

1 5

<210>10

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>10

Thr Ser Thr Gly Gly Tyr Asn Gly

1 5

<210>11

<211>119

<212>PRT

<213> mouse (Mus musculus)

<400>11

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

1 5 10 15

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

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr 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 Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys

85 90 95

Thr Ser Thr Gly Gly Tyr Asn Gly Tyr Phe Asn Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210>12

<211>339

<212>DNA

<213> mouse (Mus musculus)

<400>12

gatattgtga tgactcagac acctagctct ctggcaatga gcgtgggtca gaaggtgact 60

atgaactgta agtcaagcca gtccctgttg aactctagca accaaaagaa ctatctggcc 120

tggtaccagc agaagcccgg tcagagccca aaattgctga tatatttcgc cagtacacgg 180

gaatccggcg ttccagaccg ttttattggt agcgggtccg gcacagactt taccctgact 240

attagctccg tgcaagcaga ggatctggcc gattatttct gtcagcagca gtataacaca 300

cctctgacct tcggtgccgg taccaagctg gagattaag 339

<210>13

<211>357

<212>DNA

<213> mouse (Mus musculus)

<400>13

cagattcaac tggtgcagag cggacctgaa ctgaaaaagc caggagaaac cgtaaagttg 60

tcctgcaagg cctccggcta cacattcact aatagcggga tgaactgggt caaacaggca 120

ccaggaaagg gattgaagtg gatgggctgg atcaacacat acactggtga acctacttac 180

gccgatgact ttaaaggacg gttcgctttc tctctcgaga cttccgctag tacagcttac 240

ctgcagctga acaacttgcg agccgaggat actgggattt attactgtac ttcaacagga 300

ggctacaatg gatactttaa ctactggggg cagggcacac ttgtcactgt gagtgca 357

<210>14

<211>6

<212>PRT

<213> mouse (Mus musculus)

<400>14

Glu Asn Ile Tyr Ser Tyr

1 5

<210>15

<211>3

<212>PRT

<213> mouse (Mus musculus)

<400>15

Asn Ala Lys

1

<210>16

<211>9

<212>PRT

<213> mouse (Mus musculus)

<400>16

Gln His His Tyr Gly Thr Pro Trp Thr

1 5

<210>17

<211>107

<212>PRT

<213> mouse (Mus musculus)

<400>17

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

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

50 55 60

Leu Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>18

<211>10

<212>PRT

<213> mouse (Mus musculus)

<400>18

Gly Phe Ser Leu Ser Thr Ser Gly Met Gly

1 5 10

<210>19

<211>7

<212>PRT

<213> mouse (Mus musculus)

<400>19

Ile Trp Trp Asp Asp Asp Lys

1 5

<210>20

<211>12

<212>PRT

<213> mouse (Mus musculus)

<400>20

Ala Arg Val Gly Gly Leu Thr Thr Asn Phe Asp Val

1 5 10

<210>21

<211>120

<212>PRT

<213> mouse (Mus musculus)

<400>21

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala Gln Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Ser Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Val GlyGly Leu Thr Thr Asn Phe Asp Val Trp Gly Thr

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>22

<211>321

<212>DNA

<213> mouse (Mus musculus)

<400>22

gatattcaga tgacacagtc tcctgcttct cttagtgctt ccgtgggaga aaccgttact 60

attacttgca gagcatctga gaatatttat tcctatctcg cctggtacca gcagaaacag 120

ggaaagagcc ctcaattgtt ggtgtacaac gctaagaccc tggccgaagg agtgccctcc 180

agatttagcg ggcttggttc cggcacacag tttagcttga aaattaactc actccagcca 240

gaggatttcg ggaactacta ctgtcagcac cattatggaa caccttggac cttcggcggc 300

ggtaccaagc ttgagattaa a 321

<210>23

<211>360

<212>DNA

<213> mouse (Mus musculus)

<400>23

caggtcacat tgaaagaaag tggccctggt attctccagc catcacagac actgtctttg 60

acctgttcct tctccggctt tagtctgtcc acctcaggca tgggagtagg ctggattcgc 120

cagccttctg gaaagggtct tgagtggctt gcccaaatat ggtgggatga tgataaatat 180

tacaacactg ccctgaagag ccgtcttacc attagcaaag acacaagcaa gaatcaggtc 240

tttctgaaga tcgcctccgt cgacacagcc gactccgcta cttactactg cgctcgagtg 300

gggggcttga ccaccaactt cgatgtctgg ggaacaggga ccactgtaac tgtgtctagt 360

Claims

1. An NMP22 antibody pair characterized by: comprises a monoclonal antibody AntiNMP22_ N combined with the amino acid sequence of the N-terminal epitope of NMP22 shown as SEQ ID NO. 2 and a monoclonal antibody AntiNMP22_ C combined with the amino acid sequence of the C-terminal epitope of NMP22 shown as SEQ ID NO. 3,

the AntiNMP22_ N comprises a light chain variable region of an amino acid sequence shown as SEQ ID NO. 7 and a heavy chain variable region of an amino acid sequence shown as SEQ ID NO. 11, and the AntiNMP22_ C comprises a light chain variable region of an amino acid sequence shown as SEQ ID NO. 17 and a heavy chain variable region of an amino acid sequence shown as SEQ ID NO. 21.

2. The pair of NMP22 antibodies according to claim 1, characterized in that: the light chain of the AntiNMP22_ N comprises three amino acid sequences of LCDR1 shown as SEQ ID NO. 4, LCDR2 shown as SEQ ID NO. 5 and LCDR3 shown as SEQ ID NO. 6, the heavy chain of the AntiNMP22_ N comprises three amino acid sequences of HCDR1 shown as SEQ ID NO. 8, HCDR2 shown as SEQ ID NO. 9 and HCDR3 shown as SEQ ID NO. 10,

3. The pair of NMP22 antibodies according to claim 1, characterized in that: the AntiNMP22_ N and the AntiNMP22_ C are IgG type monoclonal antibodies, and the AntiNMP22_ N and the AntiNMP22_ C can be converted into NMP22 binding fragments Fab, F (ab ') 2, Fab', scFv and di-scFv.

4. The pair of NMP22 antibodies according to claim 1, characterized in that: the AntiNMP22_ N is a capture antibody, and the AntiNMP22_ C antibody is a detection antibody.

5. The pair of NMP22 antibodies according to claim 1 or 4, characterized in that: the detection label of the AntiNMP22_ C is enzyme, fluorescent group or radioactive isotope.

6. The pair of NMP22 antibodies according to claim 1, characterized in that: the AntiNMP22_ N and the AntiNMP22_ C can be expressed by recombination with detection marker proteins, wherein the detection marker proteins are horseradish peroxidase, alkaline phosphatase, luciferase and beta-galactosidase.

7. The pair of NMP22 antibodies according to claim 1 or 6, characterized in that: the amount of NMP22 in biological samples was detected by immunological detection methods including enzyme linked immunosorbent assay (ELISA), chemiluminescent detection, western blot detection and Immunohistochemical (IHC) detection.

8. A chemiluminescence detection kit is characterized in that: the antibody pair containing the AntiNMP22_ N combined with the N-terminal epitope of NMP22 and the AntiNMP22_ C combined with the C-terminal epitope of MP22 specifically recognizes the AntiNMP22_ C by a chemiluminescence detection method and is used for detecting the content of NMP 22.