CN111825765A - Paired antibody for detecting content of G17 in serum and application thereof - Google Patents

Abstract

The invention discloses a paired antibody for detecting the content of G17 in serum and application thereof, which comprises a monoclonal antibody AntiG17_ N combined with an amino acid sequence shown as SEQ ID NO. 2 of an N-terminal epitope of G17 and a monoclonal antibody AntiG17_ C combined with an amino acid sequence shown as SEQ ID NO. 3 of a C-terminal epitope of G17, specifically recognizes the AntiG17_ N, AntiG17_ C by adopting a chemiluminescence detection method, and detects the content of G17. The G17 antigen is creatively divided into two sequences of an N end and a C end to immunize mice respectively, and antibodies with high specificity and high sensitivity are screened for anti G17_ N and anti G17_ C and used for detecting the content of gastrin 17 in serum, so that the damage condition of the gastric mucosa of a patient is judged, and the method has important significance for guiding the safety of medication clinically.

Description

Paired antibody for detecting content of G17 in serum and application thereof

Technical Field

The invention relates to the field of stomach disease diagnosis, in particular to a paired antibody for detecting the content of G17 in serum and application thereof.

Background

Gastrin is a gastrointestinal hormone secreted mainly by G cells of the antrum and duodenum and plays an important role in regulating the function of the digestive tract and maintaining its structural integrity. In humans, more than 95% of the biologically active gastrins are alpha-amidated gastrins, which contain mainly two isomers, G-17 and G-34, of which 80% to 90% are G-17. G-17 is secreted only by G cells in the antrum of the stomach, and therefore G-17 is an important index reflecting the damage of gastric mucosa.

According to the literature, "serological evaluation of pepsinogen, gastrin-17 and Hp-IgG antibody on gastric mucosa conditions of patients with gastric cancer and atrophic gastritis", research on noninvasive serological screening method for patients with gastric cancer at high risk by applying serum PG I, G-17, PG I/PG II ratio and Hp infection conditions and evaluating gastric mucosa conditions of patients with early gastric cancer and atrophic gastritis is known.

The screening method comprises the following steps: 65 patients with gastric cancer are diagnosed by endoscope and histopathology, 70 patients with atrophic gastritis and 50 patients with normal control group are diagnosed by endoscope. Each group of sera PG I, PG II, G-17 and Hp-IgG was tested by ELISA. The results are as follows: when the stomach body is atrophic, the level of PG I in the serum of the stomach cancer group and the ratio of PG I to PG II are reduced, and the level and the ratio of PG I to PG II are obviously different (P is less than 0.01) compared with the contrast group and also obviously different (P is less than 0.05) compared with the gastritis group. When the gastric cancer is multifocal, the level of PG I in serum and the ratio of PG I to PG II in a gastric cancer group are obviously reduced, the gastric cancer group is very different from a normal control group (P is less than 0.01 and P is less than 0.001), the gastric cancer group is also obviously different from a gastritis group (P is less than 0.05 and P is less than 0.01), the level of G-17 in serum is obviously reduced, the gastric cancer group is statistically significant compared with the normal control group and the gastritis group (P is less than 0.001 and P is less than 0.05), and the ratio of multifocal atrophic lesions in gastric cancer is obviously higher than that of the gastritis group. Hp infection, however, did not affect the levels of PG I and G17 in the serum of gastric cancer patients. The conclusion is that: the low levels of PG I, PG I/PG II ratio and G-17 indicate that the patient may have high-risk multifocal atrophic lesions with gastric cancer, and the detection of serum PGs and G-17 can be used as a noninvasive examination method for gastric mucosa atrophy.

Disclosure of Invention

In view of the above-mentioned drawbacks and deficiencies of the prior art, it is desirable to provide a conjugated antibody for detecting the content of G17 in serum and the application thereof.

According to the technical scheme provided by the embodiment of the application, the paired antibodies for detecting the content of G17 in serum comprise a monoclonal antibody AntiG17_ N combined with an amino acid sequence shown as SEQ ID NO. 2 of an N-terminal epitope of G17 and a monoclonal antibody AntiG17_ C combined with an amino acid sequence shown as SEQ ID NO. 3 of a C-terminal epitope of G17,

the AntiG17_ 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 AntiG17_ 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 AntiG17_ N comprises three amino acid sequences shown as a light chain LCDR1 (SEQ ID NO: 4), a LCDR2 (SEQ ID NO: 5) and a LCDR3 (SEQ ID NO: 6), a heavy chain HCDR1 (SEQ ID NO: 8), an HCDR2 (SEQ ID NO: 9) and an HCDR3 (SEQ ID NO: 10), the AntiG17_ C comprises three amino acid sequences shown as a light chain LCDR1 (SEQ ID NO: 14), an LCDR2 (SEQ ID NO: 15), an LCDR3 (SEQ ID NO: 16), a heavy chain HCDR1 (SEQ ID NO: 18), an HCDR2 (SEQ ID NO: 19) and an HCDR3 (SEQ ID NO: 20).

In the present invention, the antibody partner may be converted to a G17 binding fragment Fab, F (ab ') 2, Fab', scFv, di-scFv.

In the invention, the anti 17_ N and the anti 17_ C are IgG type monoclonal antibodies, the anti 17_ N is used as a capture antibody, the anti 17_ C is used as a detection antibody, and the detection label of the anti 17_ C is enzyme, a fluorescent group or a radioactive isotope.

In the invention, the paired antibodies can be recombined and expressed with detection marker proteins, and the detection marker proteins are horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, glucose oxidase, lysozyme and malate dehydrogenase.

In the invention, the content of the paired antibodies is detected by an immunological detection method, and the immunological detection method comprises enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection, western blot detection, Immunohistochemistry (IHC) and immunochromatography detection.

A chemiluminescence detection kit comprises AntiG17_ N combined with N-terminal epitope of G17 and AntiG17_ C combined with C-terminal epitope of G17, specifically recognizes AntiG17_ C by a chemiluminescence detection method, and is used for detecting the content of G17.

To sum up, the beneficial effect of this application: the G17 antigen is creatively divided into two sequences with the N end shown as SEQ ID NO. 2 and the C end shown as SEQ ID NO. 3 to immunize mice respectively, and antibodies with high specificity and high sensitivity are screened for anti G17_ N and anti G17_ C and are used for detecting the content of gastrin 17 in serum, so that the damage condition of the gastric mucosa of a patient is judged, and the method has important significance for guiding the safety of medication clinically.

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 AntiG17_ N protein of 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 AntiG17_ 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 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.

A paired antibody for detecting the content of G17 in serum comprises a monoclonal antibody AntiG17_ N combined with an amino acid sequence shown as SEQ ID NO. 2 of an N-terminal epitope of G17 and a monoclonal antibody AntiG17_ C combined with an amino acid sequence shown as SEQ ID NO. 3 of a C-terminal epitope of G17, wherein the AntiG17_ 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 AntiG17_ 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. The AntiG17_ N comprises three amino acid sequences shown as a light chain LCDR1 SEQ ID NO. 4, a LCDR2 SEQ ID NO. 5 and a LCDR3 SEQ ID NO. 6, a heavy chain HCDR1 SEQ ID NO. 8, a HCDR2 SEQ ID NO. 9 and a HCDR3 SEQ ID NO. 10, the AntiG17_ C comprises three amino acid sequences shown as a light chain LCDR1 SEQ ID NO. 14, a LCDR2 SEQ ID NO. 15, a LCDR3 SEQ ID NO. 16, a heavy chain HCDR1 SEQ ID NO. 18, a HCDR2 SEQ ID NO. 19 and a HCDR3 SEQ ID NO. 20. The companion antibody can be converted to the G17 binding fragment Fab, F (ab ') 2, Fab', scFv, di-scFv. The anti 17_ N and the anti 17_ C are IgG monoclonal antibodies, the anti 17_ N is used as a capture antibody, the anti 17_ C is used as a detection antibody, and the detection label of the anti 17_ C is an enzyme, a fluorescent group or a radioactive isotope. The conjugated antibody can be recombined and expressed with detection marker protein, and the detection marker protein is horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, glucose oxidase, lysozyme and malate dehydrogenase. The content of the paired antibodies is detected by an immunological detection method, and the immunological detection method comprises enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection, western blot detection and Immunohistochemistry (IHC) and immunochromatography detection.

A chemiluminescence detection kit comprises AntiG17_ N combined with an N-terminal epitope of G17 and AntiG17_ C combined with a C-terminal epitope of G17, specifically recognizes AntiG17_ C by a chemiluminescence detection method, and is used for detecting the content of G17.

Example 1

Preparation of monoclonal antibody AntiG17_ N and monoclonal antibody AntiG17_ C

a) The N-terminal epitope (SEQ ID NO:2) of G17 and the C-terminal epitope (SEQ ID NO:3) of G17 were synthesized, respectively, and mice were immunized after coupling with KLH protein, respectively, and were 8-week-old female Balb/C mice.

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

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 by using a 10ml pipette (stopping the PEG effect); standing at 20-27 deg.C for 10 min.

f) Centrifuge at 1000r/min for 5 min and discard 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 AntiG17_ N and monoclonal antibody AntiG17_ C

The cloned antibodies obtained in example 1 were screened for the N-terminal antigen (SEQ ID NO:2) and the C-terminal antigen (SEQ ID NO:3), respectively. Finally, antibody pairs AntiG17_ N and AntiG17_ C with optimal activity and pairing, respectively, were obtained, wherein AntiG17_ N was able to specifically bind to the N-terminal antigen of G17(SEQ ID NO:2) and not to the C-terminal antigen of G17(SEQ ID NO: 3). AntiG17_ C specifically binds to the C-terminal antigen of G17(SEQ ID NO:3) but not to the N-terminal antigen of G17(SEQ ID NO: 2).

G17 whole antigen (SEQ ID NO:1) was coated and verified by chemiluminescence.

The N-terminal antigen of G17(SEQ ID NO:2) was coated and detected by chemiluminescence.

The C-terminal antigen of G17(SEQ ID NO:3) was coated and detected by chemiluminescence.

Respectively coating the G17 holoantigen (SEQ ID NO:1), the N-terminal antigen (SEQ ID NO:2) of G17 and the C-terminal antigen (SEQ ID NO:3) of G17, adding different volumes of AntiG17_ N and AntiG17_ C, adding a secondary antibody for reaction, finally adding a substrate solution, and detecting the light-emitting value, wherein the results are as follows:

TABLE 1

	Whole antigen (SEQ ID NO:1)	N-terminal antigen (SEQ ID NO:2)	C-terminal antigen (SEQ ID NO:3)
				Blank group	7284	7416	7441
AntiG17_N 2ul	127823	127347	7284
				AntiG17_N 10ul	571879	519624	7684
AntiG17_N 50ul	2704866	2602532	7508
				AntiG17_C 10ul	127896	7367	120010
AntiG17_C 20ul	586989	7430	525512
				AntiG17_C 50ul	2739263	7369	2580593

From table 1, it was confirmed that anti 17_ N in the antibody pair of the present invention recognizes the N-terminal antigen and does not recognize the C-terminal antigen. The antibody pair of the invention has the ability of recognizing the C-terminal antigen and the ability of recognizing the N-terminal antigen by AntiG17_ C.

Example 3

Recombinant expression of the N-terminal antibody AntiG17_ N of G17 and the C-terminal antibody AntiG17_ C of G17

a. N-terminal antibody AntiG17_ N of recombinant expression G17

After sequencing the hybridomas of antibody 17_ 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 sequence (SEQ ID NO:11) of antibody 17_ 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 (shown in FIG. 2). The variable region of the heavy chain was cloned into pcDNA3.1-HFc vector to obtain pcDNA3.1-NH-HFc (shown in 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 anti-G17N-terminal antibody AntiG17_ N (as shown in 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：AntiG17_N

b. c-terminal antibody AntiG17_ C of recombinant expression G17

After sequencing the hybridomas of antibody 17_ 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 sequence (SEQ ID NO:21) of antibody 17_ C were obtained. After optimizing the sequence, the light chain variable region was cloned into pcDNA3.1-LFc vector to obtain pcDNA3.1-CL-LFc (shown in FIG. 5). The variable region of the heavy chain was cloned into pcDNA3.1-HFc vector to obtain pcDNA3.1-CH-HFc (shown in 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. The pcDNA3.1-CL-LFc and pcDNA3.1-CH-HFc were co-transfected into CHO-S cells at a ratio of 1:1 to recombinantly express the anti-G17C-terminal antibody AntiG17_ C (as shown in 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：AntiG17_C

example 4

Preparation and verification of paired antibody participating chemiluminescence kit

According to the existing production process of a chemiluminescence method diagnostic reagent, the anti 17_ C of the invention is used as a capture antibody to be coated to be configured into a G-17 coating plate, the anti 17_ N of the invention is used as a binding antibody to be labeled to be configured into a G-17 enzyme conjugate, and the G-17 antigen is configured into a series of calibrators (0pmol/L, 1pmol/L, 4pmol/L, 16pmol/L, 64pmol/L and 256pmol/L) to form a complete kit and carry out a series of verifications.

The performance verification index of the kit prepared from the antibody is formulated by 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 G-17 kit manufacturers in the market.

1. Performance index

1.1 minimum detection Limit

Should not be higher than 0.6 pmol/L.

1.2 dose-response Curve Linearity

The dose-response curve linear correlation coefficient (r) should be not less than 0.9900 within a linear interval of 1 to 256 pmol/L.

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%.

1.5 specificity

5.0pmol/L human cholecystokinin (CCK), the apparent value should not be greater than 0.5 pmol/L.

2. Inspection method

2.1 minimum detection Limit

The relative luminous intensity of a 20-hole zero calibrator (0pg/ml) is measured in parallel, the mean value and the standard deviation SD of the luminous value are calculated, the concentration value corresponding to the luminous value of the mean value plus 2 xSD is calculated by using a dose-response curve, namely the lowest detection limit, and the result meets the requirement of 1.1.

2.2 dose-response Curve linearity

The reference substance of the double-well assay kit (S0-S5, the concentration is 0pmol/L, 1pmol/L, 4pmol/L, 16pmol/L, 64pmol/L and 256pmol/L in sequence), and the dose-response curve of the kit is fitted by a double logarithm method, and the linear correlation coefficient r should meet the requirement of 1.2.

2.3 accuracy

G-17 antigen was prepared as a standard solution (180pmol/L) in a volume ratio of 1: 9 was added to a low value sample (3.82-5.39pmol/L) of known concentration, and the assay was carried out on the kit, and the recovery rate R was calculated according to equation (1) and the result was in accordance with the 1.3 specification.

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 parallel measurement of 10 wells of each of the quality control Q1 and the quality control Q2 according to the formula, and the result was in accordance with 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

2.5 specificity

Human cholecystokinin (CCK) is added into normal human serum to prepare a CCK sample containing 5.0pmol/L for detection, and the apparent value of the CCK sample meets the requirement of 1.5.

3. Experimental configuration

3.1 calibrator configuration

The G-17 antigen was diluted in six gradients (0pmol/L, 1pmol/L, 4pmol/L, 16pmol/L, 64pmol/L, 256pmol/L) with antigen diluent (0.5mol PBS + 1% BSA).

3.2 recovery Standard solution preparation

The G-17 antigen was diluted to 180pmol/L with an antigen diluent (0.5mol PBS + 1% BSA) as a standard solution, and 3.82 to 5.39pmol/L of normal human serum was selected as a low-value sample.

3.3 quality control product configuration

The G-17 antigen was diluted with antigen diluent (0.5mol PBS + 1% BSA) to Q1 (3.33. + -. 1.05pmol/L) and Q2 (184.8. + -. 44.65 pmol/L).

3.4 specific sample configuration

Human cholecystokinin (CCK) was prepared in 5.0pmol/L CCK sample using a diluent (0.5mol PBS + 1% BSA).

4. Sample application operation

(1) Respectively adding 100 μ l of the 3.1-3.4G-17 calibrator, the recovery sample, the quality control sample and the specific sample into corresponding coated plate wells, and oscillating 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 μ L G-17 enzyme conjugate was added to each well and the shaker shaken for 30 seconds

(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 Beijing Lidmann Biochemical Co., Ltd.) 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 0.272pmol/l, which is not higher than 0.6pmol/l, and meets the criteria.

5.2 dose-response Curve Linearity

TABLE 3

	First measurement	Second measurement	The third measurement
				Linear correlation coefficient R	0.9999	0.9994	0.9996

As can be seen from Table 3, FIG. 9, FIG. 10 and FIG. 11, the linear correlation coefficient r is greater than 0.9900 and meets the index when the G-17 antigen curve in the concentration range of 0-256pmol/L is measured three times continuously.

5.3 accuracy

TABLE 4

CS(pmol/L)	C0(pmol/L)	Sample C luminescence value	C(pmol/L)	Recovery (%)
					130	3.5	616883	16.444	102.26

As can be seen from Table 4, the recovery rate detected with the kit was in the range of 0.85-1.15, which met the criteria.

5.4 precision

TABLE 5

As can be seen from Table 5, the results of three consecutive parallel determinations of QC1 QC2 were no more than 10% and met the index.

5.5 specificity

TABLE 6

Item	Luminous value	Concentration value (pg/mL)
			Human cholecystokinin (5.0pmol/L)	8245	0.293

As can be seen from Table 6, human cholecystokinin (5.0pmol/L) was detected at 0.293pmol/L and not more than 0.5pmol/L, which was satisfactory.

From the above, the dose-response curve of the G-17 kit prepared by the invention has performance indexes such as accuracy, uniformity and the like which all meet the technical requirements.

Example 5

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

Randomly selecting 40 parts of human serum specimen, preparing a kit by using the antibody of the invention and detecting the serum by using the conventional G-17 kit (Shandongda) in the market, and comparing the correlation

A comparison of 40 human serum samples was as follows:

TABLE 7

As is clear from Table 7 and FIG. 9, the correlation R2 between the concentration results of the present invention and the concentration results of the existing kits on the market is 0.9962.

The detection result of the clinical sample is consistent with the detection result of the existing chemiluminescence method detection reagent (Shandongda) in the market.

Example 6

The sequence of G17 is shown in SEQ ID NO 1. Because the antigen of G17 is small, there is a great difficulty in developing antibody pairs. Meanwhile, the developed antibody pair has clinical application value and is more difficult. The G17 antigen is creatively divided into two sequences of N terminal (SEQ ID NO:2) and C terminal (SEQ ID NO: 3). And the two sequences are used for immunizing mice respectively, and antibody pairs of AntiG17_ N and AntiG17_ C with high specificity and high sensitivity are screened.

The present invention provides the core sequence of the G17 antibody pair (variable region of antibody to heavy and light chains). Wherein SEQ ID NO 4, 5, 6 in the light chain sequence and 8, 9, 10 in the heavy chain sequence of AntiG17_ N are the key positions for the binding of the N-terminal epitope (SEQ ID NO:2) of the G17 antigen. 14, 15, 16 in the light chain sequence and 18, 19, 20 in the heavy chain sequence of AntiG17_ C are the key positions for the binding of the G17 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 light chain of antibody AntiG17_ N in the antibody pair of the present invention comprises all of SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6. The heavy chain comprises all of SEQ ID NO 8, 9, 10.

The light chain of antibody AntiG17_ C in the antibody pair of the invention comprises all of SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16. The heavy chain comprises all of SEQ ID NO 18, 19, 20.

Antibody AntiG17_ N in the antibody pair of the invention comprises the light chain variable region comprising the amino acid sequence according to SEQ ID NO. 7 and the heavy chain variable region comprising the amino acid sequence according to SEQ ID NO. 11.

Antibody AntiG17_ C in the antibody pair of the invention comprises the light chain variable region comprising the amino acid sequence according to SEQ ID NO 17 and the heavy chain variable region comprising the amino acid sequence according to SEQ ID NO 21.

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

Antibody AntiG17_ N in the antibody pairs of the invention is a capture antibody and AntiG17_ C is a labeled antibody, which may be further conjugated to a detectable label.

The antibody of antibody pair of the invention, antibody AntiG17_ C, is detectably labeled with a semienzyme or enzyme or a fluorophore or radioisotope.

The antibodies of the invention are useful for detecting the presence of G17(SEQ ID NO:1) in a sample that is a biological sample, preferably serum.

The antibody pair of the present invention was used to detect the presence of G17(SEQ ID NO:1) by a sandwich immunological method. The present implementation class selects chemiluminescence methodology for detection. Therefore, the content of G17 in the biological sample can be accurately detected.

The antibody of the invention detects the existence and expression of G17 by a chemical light-emitting method for AntiG17_ N and AntiG17_ C.

The samples used in the in vitro methods of the invention are derived from subjects suffering from or at risk of: gastritis, gastric ulcer and gastric cancer.

The present invention relates to the use of antibodies of the invention against AntiG17_ N and AntiG17_ C in detecting G17 expression in a sample as disclosed above.

The invention provides the amino acid sequences of antibodies to the variable regions of AntiG17_ N and AntiG17_ C.

The present invention provides expression vectors pcdna3.1-AntiG17_ NL and pcdna3.1-AntiG17_ NH for expression of AntiG17 comprising heavy and light chain polynucleotides encoding the antibody AntiG17_ N of the present invention.

The present invention provides expression vectors pcdna3.1-AntiG17_ CL and pcdna3.1-AntiG17_ CH for expressing AntiG17, comprising heavy and light chain polynucleotides encoding the antibody AntiG17_ C of the present invention.

The invention provides an expression vector for producing the antibody pair AntiG17_ N and AntiG17_ 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 of diagnosing a gastric disease comprising the steps of: the expression of G17 in patient samples was detected using the chemiluminescence detection method using the antibody pairs AntiG17_ N and AntiG17_ C of the invention as disclosed above. If the content of G17 is not within the normal range, the risk of gastritis and gastric 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 embodiments in which the above-described technical features are combined in particular, and the invention also covers other embodiments in which the above-described technical features or their equivalent are combined arbitrarily without departing from the scope of the invention. For example, the above-mentioned features and the technical features (but not limited to) having similar functions disclosed in this application are mutually replaced to form the technical solution.

Sequence listing

<110> corridor sky light biotechnology Limited

<120> paired antibody for detecting G17 content in serum and application thereof

<160>23

<170>SIPOSequenceListing 1.0

<210>1

<211>17

<212>PRT

<213> mouse (Mus musculus)

<400>1

Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp

1 510 15

Phe

<210>2

<211>6

<212>PRT

<213> mouse (Mus musculus)

<400>2

Gly Pro Trp Leu Glu Glu

1 5

<210>3

<211>6

<212>PRT

<213> mouse (Mus musculus)

<400>3

Tyr Gly Trp Met Asp Phe

1 5

<210>4

<211>5

<212>PRT

<213> mouse (Mus musculus)

<400>4

Ser Ser Val Ser Tyr

1 5

<210>5

<211>3

<212>PRT

<213> mouse (Mus musculus)

<400>5

Asp Thr Ser

1

<210>6

<211>10

<212>PRT

<213> mouse (Mus musculus)

<400>6

Gln Gln Trp Ser Arg His Pro Pro Ile Thr

1 5 10

<210>7

<211>107

<212>PRT

<213> mouse (Mus musculus)

<400>7

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Val Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg His Pro Pro Ile

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210>8

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>8

Gly Phe Thr Phe Ser Asn Ala Trp

1 5

<210>9

<211>10

<212>PRT

<213> mouse (Mus musculus)

<400>9

Ile Arg Leu Lys Ala Asn Asn His Ala Thr

1 5 10

<210>10

<211>6

<212>PRT

<213> mouse (Mus musculus)

<400>10

Glu Ile Thr Thr Leu Tyr

1 5

<210>11

<211>123

<212>PRT

<213> mouse (Mus musculus)

<400>11

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Leu Lys Ala Asn Asn His Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Thr Arg Glu Ile Thr Thr Leu Tyr Tyr Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>12

<211>321

<212>DNA

<213> mouse (Mus musculus)

<400>12

gagatggttc ttactcagtc tcctgccatc ctgtctgcat ccccaggaga gaaggtgacc 60

atgacttgcc gcgcaagctc atcagtcagc tatatgcact ggtatcagca aagacccggc 120

agtagcccaa agccttggat ttacgatact tcaaacctgg ctagcggagt gcctgtaagg 180

ttctccggga gcggttctgg gacaagttat agcctgacca tttcaagggt tgaagtggag 240

gacgccgcca cttactactg tcagcaatgg agccgccacc ctccaattac cttcggctca 300

ggcaccaagc tggaaataaa a 321

<210>13

<211>369

<212>DNA

<213> mouse (Mus musculus)

<400>13

gaggttaagt tggaagaaag tggtggggac ttggtccagc caggtcagtc catgaaactc 60

agctgtgttg ctagcggatt cacatttagt aatgcctgga tgaattgggt acgtcaaagc 120

cctgagaaag ggttggagtg ggtggccgag ataagactga aggccaataa tcacgccact 180

cattacgcag agagtgtgaa gggccgattc acaatttccc gagacgacag caaatcttca 240

gtctatctgc agatgaacaa cctgcgagcc gaggacacag gtatctatta ctgcacacgc 300

gagatcacta cactgtacta ctacgctatg gattactggg gccaaggaac atccgtaacc 360

gtaagcagc 369

<210>14

<211>11

<212>PRT

<213> mouse (Mus musculus)

<400>14

Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr

1 5 10

<210>15

<211>3

<212>PRT

<213> mouse (Mus musculus)

<400>15

Arg Met Ser

1

<210>16

<211>9

<212>PRT

<213> mouse (Mus musculus)

<400>16

Met Gln His Leu Glu Tyr Pro Leu Thr

1 5

<210>17

<211>112

<212>PRT

<213> mouse (Mus musculus)

<400>17

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

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Asn Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Ile Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Leu Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210>18

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>18

Gly Phe Thr Leu Ser Arg Tyr Thr

1 5

<210>19

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>19

Ile Ser Ser Gly Gly Gly Asn Thr

1 5

<210>20

<211>12

<212>PRT

<213> mouse (Mus musculus)

<400>20

Ser Arg Tyr Gly Tyr Asp Gly Ala Trp Phe Ala Tyr

1 5 10

<210>21

<211>120

<212>PRT

<213> mouse (Mus musculus)

<400>21

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ile Ile Thr Ser Gly Gly Gly Gly Asn Thr Phe Tyr Pro Asp Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ser Arg Tyr Gly Tyr Asp Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210>22

<211>336

<212>DNA

<213> mouse (Mus musculus)

<400>22

gatattgtca tgacacaggc tgcccctagt gtcccagtga ctccaggaga gagcgtctcc 60

ataagttgca gatctaacaa gagtctcctc cactccaatg gtaataccta catttactgg 120

tttctgcaga gaccaggcca atctcctcaa ctcctcctgt accggatgag caacctggcc 180

tccggagtgc ctgatcgatt cagtggaagc gggtccggca cagcctttac cctgcgaatc 240

tccagggtgg aggcagagga tgtgggcgtc tactattgta tgcagcacct cgaatatcct 300

ctcacatttg gtgcaggcac caagctcgag ctcaag 336

<210>23

<211>360

<212>DNA

<213> mouse (Mus musculus)

<400>23

gaggtgatgc tcgtagagtc aggaggagga cttgtcaaac ctggtggtag tctgaagctt 60

agctgcgcag catccggctt tactctcagt aggtatacta tgagttgggt ccgtcagacc 120

cctgagaagc ggctcgagtg ggtcgctatc ataacatccg ggggcggagg aaacactttt 180

taccccgaca gcgtgaaggg tcggtttacc atcagtcgtg ataatgccaa aaacaatctt 240

tatttgcaga tgagctctct tcgtagcgag gataccgccc tttattattg ttcacgatat 300

gggtatgatg gcgcctggtt tgcttattgg gggcagggga cactggtcac tgtgagtgct 360

Claims (7)

1. A paired antibody for detecting the content of G17 in serum, which is characterized in that: comprises a monoclonal antibody AntiG17_ N combined with the amino acid sequence shown as SEQ ID NO. 2 of the N-terminal epitope of G17 and a monoclonal antibody AntiG17_ C combined with the amino acid sequence shown as SEQ ID NO. 3 of the C-terminal epitope of G17,

the AntiG17_ 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 AntiG17_ 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 paired antibody of claim 1, which is used for detecting the content of G17 in serum, and is characterized in that:

the AntiG17_ N comprises three amino acid sequences shown as a light chain LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5) and LCDR3 (SEQ ID NO: 6), a heavy chain HCDR1 (SEQ ID NO: 8), HCDR2 (SEQ ID NO: 9) and HCDR3 (SEQ ID NO: 10),

the AntiG17_ C comprises three amino acid sequences shown as a light chain LCDR1 SEQ ID NO. 14, a LCDR2 SEQ ID NO. 15 and a LCDR3 SEQ ID NO. 16, a heavy chain HCDR1 SEQ ID NO. 18, an HCDR2 SEQ ID NO. 19 and an HCDR3 SEQ ID NO. 20.

3. The paired antibody of claim 1, which is used for detecting the content of G17 in serum, and is characterized in that: the companion antibody can be converted to the G17 binding fragment Fab, F (ab ') 2, Fab', scFv, di-scFv.

4. The paired antibody of claim 1, which is used for detecting the content of G17 in serum, and is characterized in that: the anti 17_ N and the anti 17_ C are IgG monoclonal antibodies, the anti 17_ N is used as a capture antibody, the anti 17_ C is used as a detection antibody, and the detection label of the anti 17_ C is an enzyme, a fluorescent group or a radioactive isotope.

5. The paired antibody of claim 1, which is used for detecting the content of G17 in serum, and is characterized in that: the conjugated antibody can be recombined and expressed with detection marker protein, and the detection marker protein is horseradish peroxidase, alkaline phosphatase, luciferase, beta-galactosidase, glucose oxidase, lysozyme and malate dehydrogenase.

6. The paired antibody of claim 1, which is used for detecting the content of G17 in serum, and is characterized in that: the content of the paired antibodies is detected by an immunological detection method, and the immunological detection method comprises enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection, western blot detection and Immunohistochemistry (IHC) and immunochromatography detection.

7. A chemiluminescence detection kit is characterized in that: contains the AntiG17_ N combined with the N-terminal epitope of G17 and the AntiG17_ C combined with the C-terminal epitope of G17, specifically recognizes the AntiG17_ C by a chemiluminescence detection method, and is used for detecting the content of G17.

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