CN113817063A - Anti-human abnormal prothrombin antibody and application thereof - Google Patents

Abstract

The invention belongs to the field of antibodies, and particularly relates to an anti-human abnormal prothrombin antibody and application of the antibody in preparation of a hepatocellular carcinoma detection kit. The anti-human abnormal prothrombin antibody can be specifically combined with human abnormal prothrombin, can further avoid the interference from prothrombin in serum or plasma from the raw material level, can also further avoid the interference from other species of abnormal prothrombin in an animal serum product, avoids related risks from the source, reduces the difficulty of reagent development, and has good application prospect.

Description

Anti-human abnormal prothrombin antibody and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to an anti-human abnormal prothrombin antibody and application thereof.

Background

Normal prothrombin is produced by carboxylating gamma-carboxyglutamic acid (Glu) residues at positions 6, 7, 14, 16, 19, 20, 25, 26, 29 and 32 of the N-terminal of the domain to gamma-carboxyglutamic acid (Gla) in the presence of gamma glutamyl carboxylase and related coenzymes which are mainly dependent on vitamin K in hepatocytes, and thus is converted into active normal prothrombin, wherein one or more Glu residues at any of the above sites are not fully carboxylated and may form abnormal prothrombin (Des-gamma-carboxypropylthrombin, hereinafter referred to as DCP) to lose the blood coagulation function. This N-terminus is currently referred to as the Glu-Gla region, and except for this region, normal prothrombin and abnormal prothrombin are not structurally different and consist of the prothrombin fragment F1, prothrombin fragment F2 and the C-terminal thrombin region. DCP, also known as vitamin K deficient or antagonist-II induced Protein (Protein induced by vitamin K absence-II, PIVKA-II). The content of DCP is increased by vitamin K deficiency, warfarin treatment and hepatocellular carcinoma (HCC) patients, so that DCP can be used as a diagnostic marker of HCC. In recent years, with the development of more research works, the clinical application value of DCP in HCC is generally recognized, and DCP is included in various authority guidelines and recommended to be used for screening of high risk groups, auxiliary diagnosis of liver cancer, monitoring of treatment effect, and as a prediction tool for prognosis and recurrence.

With the approval of the clinical application value of DCP, various immunodetection methods are available, and the sensitivity and specificity of the immunodetection method are of no doubt to the importance of DCP in clinical diagnosis. When the adopted antibody and other antigens or substances except the DCP in the sample have non-specific interaction, the possibility of false positive diagnosis is increased, and the risk that an individual cannot be accurately diagnosed is increased, so whether the antibody adopted in the detection method is specifically combined with the DCP in the human sample or not is the key for ensuring the sensitivity and specificity of the immunoassay method.

In Japanese patent laid-open No. 60-60557 and Japanese patent laid-open No. 5-249108, respectively, there are reported immunoassay methods using rabbit polyclonal antibodies that specifically recognize DCP monoclonal antibody MU-3(17-27aa) as a coating, which avoids the sample pretreatment step, simplifies the flow, makes the DCP detection method more practical than the early radioimmunoassay, and anti-prothrombin, which adjusts the specifically recognized DCP monoclonal antibody MU-3(17-27aa) as a marker, reduces prothrombin interference in the sample to obtain more stable detection data; in the immunoassay method reported in patent US9120862B2, a monoclonal antibody 3C10(13-27aa) specifically recognizing DCP and an anti-prothrombin antibody 6H6(1-13aa) were used; the immunoassay method reported in patent CN 104520710B adopts an antibody MU-3(17-27aa) for specifically recognizing DCP, an antibody hPTN7-2 for specifically recognizing prothrombin F1 fragment and an antibody 20B8 for specifically recognizing prothrombin F2, and establishes a detection system with high serum/plasma correlation by the combined application of three antibodies, particularly the proportional application of F1 and F2 fragment antibodies; among other publications, H.Kinukawa et al.clinical Biochemistry xxx (2015) xxx-xxx discloses a DCP immunodetection method, which adopts a 3C10(13-27aa) antibody specifically recognizing DCP and combines another antithrombin antibody MCA1-8(33-46aa) to construct a chemiluminescence detection reagent of DCP.

The current research mainly centers on an application method for detecting DCP or a corresponding antibody by matching an antibody for recognizing a specific epitope of DCP and an antibody for prothrombin, and no report on specific detection of a human DCP antibody exists.

Disclosure of Invention

In view of the above, the present invention provides a novel anti-human abnormal prothrombin antibody with an amino acid sequence, a vector containing a nucleic acid encoding the antibody, a transgenic cell line, a recombinant bacterium, a recombinant virus and an application of the antibody in the preparation of a kit for detecting human abnormal prothrombin, which solves the problems in the background art.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides an anti-human aberrant prothrombin antibody comprising a heavy chain variable region and a light chain variable region capable of specifically binding human aberrant prothrombin, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are set forth in SEQ ID NOs 1-3, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6.

SEQ ID NO:1 GYTFTSYV

SEQ ID NO:2 IFPYNDVT

SEQ ID NO:3 ARWGGRNY

SEQ ID NO:4 QSLLDSDGKTY

SEQ ID NO:5 LVS

SEQ ID NO:6 WQGTHFPYT

Preferably, the amino acid sequence of the heavy chain variable region of the antibody is any one of the following:

(1) shown as SEQ ID NO. 7;

SEQ ID NO:7

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYIFPYNDVTEYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARWGGRNYWGQGTSVTV

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 7;

(3) an amino acid sequence of a protein which has at least 80 percent of homology with the sequence shown as SEQ ID NO. 7 and has the same function; the homology is preferably at least 85%; more preferably at least 95%.

Preferably, the amino acid sequence of the light chain variable region of the antibody is any one of the following:

(1) as shown in SEQ ID NO. 8;

SEQ ID NO:8

DVVMTQTPLTLSITIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLQISRVEAEDLGIYYCWQGTHFPYTFGGGTKLEIKR

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 8;

(3) an amino acid sequence of a protein which has at least 80 percent of homology with the sequence shown as SEQ ID NO. 8 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

The antibody of the invention can specifically bind to the human abnormal prothrombin, and the antigen recognition region of the antibody is positioned in the 31-45 amino acid region of the human abnormal prothrombin.

In a second aspect, the invention provides nucleic acids encoding such anti-human aberrant prothrombin antibodies.

In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid.

In a fourth aspect, the invention provides a transgenic cell line comprising the nucleic acid described above.

In a fifth aspect, the present invention provides a recombinant bacterium comprising the above-mentioned nucleic acid.

In a sixth aspect, the present invention provides a recombinant virus comprising the nucleic acid described above.

In a seventh aspect, the invention provides an application of the anti-human abnormal prothrombin antibody in preparing a hepatocellular carcinoma detection kit.

In an eighth aspect, the present invention provides a detection kit for hepatocellular carcinoma, which comprises the anti-human abnormal prothrombin antibody.

The detection kit for hepatocellular carcinoma may further include other reagents or materials required for detection, including but not limited to buffer, secondary antibody, etc., in addition to the anti-human abnormal prothrombin antibody.

The content of prothrombin in blood plasma can reach 0.1-0.15mg/ml, and even if the prothrombin is consumed in the process of converting the prothrombin into thrombin, about 15% of prothrombin remains in a serum product. On one hand, if the kit adopts an antibody of prothrombin when detecting the DCP in a human body, the detection result may be interfered by the prothrombin in serum or plasma, so that the detection accuracy is influenced; on the other hand, animal serum products are widely used in various links of in vitro diagnostic reagent development at present, not only antibodies using prothrombin may be interfered, but also antibodies from other species may be interfered if they do not specifically recognize human DCP, thereby affecting the accuracy of the detection result, if the non-specificity is to be avoided, other means such as system adjustment or other blocking agents are adopted to avoid or simply not use related animal serum or related products, and this limits the reagent development conditions and also increases the complexity of reagent development to some extent.

The invention has the beneficial effects that: the antihuman abnormal prothrombin can specifically identify the human antihuman abnormal prothrombin, can further avoid the interference from prothrombin in serum or plasma from the raw material level, can also further avoid the interference from abnormal prothrombin from other species in animal serum products, avoids related risks from the source, reduces the difficulty of reagent development, and has relatively wide application prospect.

Drawings

FIG. 1 is an SDS-PAGE electrophoretic identification of recombinant DCP antigen, wherein,

lane M: protein molecular weight markers;

lane 1: human DCP1-50 aa-pGEX-20T;

lane 2: human DCP1-50 aa-pColdTF.

The result shows that the target proteins are expressed in large quantity, the size of the target fragments is close to the theoretical value, and the purity is higher.

FIG. 2 is an SDS-PAGE purity identification chart of anti-human DCP monoclonal antibody, wherein

Lane M: protein molecular weight markers;

lane 1: anti-human DCP monoclonal antibodies.

FIG. 3 is a graph showing the reactivity of antigen-antibody indirect method of antigen 1 and antigen 2 with anti-human DCP monoclonal antibody in example 1.

FIG. 4 is a graph showing the correlation between a DCP prototype detection reagent and a control reagent.

Detailed Description

For better understanding of the present invention, the following embodiments and the accompanying drawings are used to describe the present invention in further detail, but those skilled in the art will appreciate that the following embodiments are not intended to limit the scope of the present invention, and any changes and modifications based on the present invention are within the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of recombinant antigen of abnormal Prothrombin

Based on the prothrombin sequence (Accession number: NM-000506.5) searched in Gene Bank, the recombinant expression clone design is carried out, and the full-length sequence is shown as SEQ ID NO: shown at 9. According to the difference between DCP and normal prothrombin, the DCP is mainly focused on the first 50 amino acids, and the reported antibody epitope is generally linear epitope, so that the expression plasmids of prothrombin 1-50aa are respectively constructed by using commercial vectors pColdTF and pGEX-20T, and the expression of target protein is carried out by using an escherichia coli expression system, because the protein cannot be subjected to gamma carboxylation modification in escherichia coli, the expression product is abnormal prothrombin, namely DCP, corresponding 1-50aa, and the specific nucleotide sequence is shown as SEQ ID NO: shown at 10.

Because the two expression vectors adopt the same enzyme cutting site, the primers are the same, primer5.0 is used, BgL II and EcoR I are used as enzyme cutting sites for primer design, and the specific sequences of the upstream primer and the downstream primer (synthesized by general biology) are respectively shown as SEQ ID NO: 11 and SEQ ID NO: 12, the amino acid sequence of the cloned target protein is shown as SEQ ID NO: shown at 13. After plasmid construction, after double verification of sequencing and small-amount expression identification, large-amount expression is carried out by using an escherichia coli expression system, bacteria are collected, ultrasonically crushed and centrifuged, protein is obtained by supernatant through affinity chromatography and purification of nickel medium (GE company), antigens expressed by pGEX-20T and pColdTF vectors are respectively named as antigen 1 and antigen 2, the result of SDS-PAGE identification is shown in figure 1, lanes 1 and 2 are respectively antigen 1 and antigen 2, the result shows that target protein can be expressed in large amount, the size of a target fragment is close to a theoretical value, and the target fragment has higher purity.

SEQ ID NO：9:

ATGGCGCACGTCCGAGGCTTGCAGCTGCCTGGCTGCCTGGCCCTGGCTGCCCTGTGTAGCCTTGTGCACAGCCAGCATGTGTTCCTGGCTCCTCAGCAAGCACGGTCGCTGCTCCAGCGGGTCCGGCGAGCCAACACCTTCTTGGAGGAGGTGCGCAAGGGCAACCTGGAGCGAGAGTGCGTGGAGGAGACGTGCAGCTACGAGGAGGCCTTCGAGGCTCTGGAGTCCTCCACGGCTACGGATGTGTTCTGGGCCAAGTACACAGCTTGTGAGACAGCGAGGACGCCTCGAGATAAGCTTGCTGCATGTCTGGAAGGTAACTGTGCTGAGGGTCTGGGTACGAACTACCGAGGGCATGTGAACATCACCCGGTCAGGCATTGAGTGCCAGCTATGGAGGAGTCGCTACCCACATAAGCCTGAAATCAACTCCACTACCCATCCTGGGGCCGACCTACAGGAGAATTTCTGCCGCAACCCCGACAGCAGCACCACGGGACCCTGGTGCTACACTACAGACCCCACCGTGAGGAGGCAGGAATGCAGCATCCCTGTCTGTGGCCAGGATCAAGTCACTGTAGCGATGACTCCACGCTCCGAAGGCTCCAGTGTGAATCTGTCACCTCCATTGGAGCAGTGTGTCCCTGATCGGGGGCAGCAGTACCAGGGGCGCCTGGCGGTGACCACACATGGGCTCCCCTGCCTGGCCTGGGCCAGCGCACAGGCCAAGGCCCTGAGCAAGCACCAGGACTTCAACTCAGCTGTGCAGCTGGTGGAGAACTTCTGCCGCAACCCAGACGGGGATGAGGAGGGCGCGTGGTGCTATGTGGCCGGGAAGCCTGGCGACTTTGGGTACTGCGACCTCAACTATTGTGAGGAGGCCGTGGAGGAGGAGACAGGAGATGGGCTGGATGAGGACTCAGACAGGGCCATCGAAGGGCGTACCGCCACCAGTGAGTACCAGACTTTCTTCAATCCGAGGACCTTTGGCTCGGGAGAGGCAGACTGTGGGCTGCGACCTCTGTTCGAGAAGAAGTCGCTGGAGGACAAAACCGAAAGAGAGCTCCTGGAATCCTACATCGACGGGCGCATTGTGGAGGGCTCGGATGCAGAGATCGGCATGTCACCTTGGCAGGTGATGCTTTTCCGGAAGAGTCCCCAGGAGCTGCTGTGTGGGGCCAGCCTCATCAGTGACCGCTGGGTCCTCACCGCCGCCCACTGCCTCCTGTACCCGCCCTGGGACAAGAACTTCACCGAGAATGACCTTCTGGTGCGCATTGGCAAGCACTCCCGCACCAGGTACGAGCGAAACATTGAAAAGATATCCATGTTGGAAAAGATCTACATCCACCCCAGGTACAACTGGCGGGAGAACCTGGACCGGGACATTGCCCTGATGAAGCTGAAGAAGCCTGTTGCCTTCAGTGACTACATTCACCCTGTGTGTCTGCCCGACAGGGAGACGGCAGCCAGCTTGCTCCAGGCTGGATACAAGGGGCGGGTGACAGGCTGGGGCAACCTGAAGGAGACGTGGACAGCCAACGTTGGTAAGGGGCAGCCCAGTGTCCTGCAGGTGGTGAACCTGCCCATTGTGGAGCGGCCGGTCTGCAAGGACTCCACCCGGATCCGCATCACTGACAACATGTTCTGTGCTGGTTACAAGCCTGATGAAGGGAAACGAGGGGATGCCTGTGAAGGTGACAGTGGGGGACCCTTTGTCATGAAGAGCCCCTTTAACAACCGCTGGTATCAAATGGGCATCGTCTCATGGGGTGAAGGCTGTGACCGGGATGGGAAATATGGCTTCTACACACATGTGTTCCGCCTGAAGAAGTGGATACAGAAGGTCATTGATCAGTTTGGAGAGTAG

SEQ ID NO：10:

ATGGCGCACGTCCGAGGCTTGCAGCTGCCTGGCTGCCTGGCCCTGGCTGCCCTGTGTAGCCTTGTGCACAGCCAGCATGTGTTCCTGGCTCCTCAGCAAGCACGGTCGCTGCTCCAGCGGGTCCGGCGAGCCAACACCTTCTTGGAGGAG

SEQ ID NO：11:

AGATCTATGGCGCACGTCCGAGGCTTGC

SEQ ID NO:12:

GAATTCCTCCTCCAAGAAGGTGTTGGCTCGCC

SEQ ID NO：13:

MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEE

EXAMPLE 2 preparation of monoclonal antibodies to abnormal Prothrombin

1. Preparation of immunogen: the immunogen was antigen 1 prepared in example 1. Antigen 1 was diluted to 0.4mg/mL with 10mmol/L PBS, and the corresponding antigen was mixed with Freund's adjuvant in equal volume at a final concentration of 200ug/mL to form a water-in-oil emulsion. Freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the booster immunization.

2. Basic immunity: selecting 6-8 weeks old BALB/c female mice to carry out subcutaneous multi-point injection immunization, wherein the immunogen injection dose is 500 mu L/mouse, the immunization interval is2 weeks, the complete immunization program is 4 needles, and separated serum is collected by an orbital blood collection method for indirect ELISA (enzyme-linked immunosorbent assay) for measuring the immune titer 2 weeks after 2, 3 and 4-needle immunization, and after the mouse serum titer reaches a platform period, the spleen immunization is carried out by 100 ug/mouse dose of antigen 1 72 hours before cell fusion.

3. Hybridoma cell selection

3.1 preparation of feeder macrophages:

(i) killing BALB/c mice with the age of about 6 weeks by introducing necks, and soaking in 75% alcohol solution for 5 min; taking out and putting into a sterile plate which is arranged on a superclean workbench in advance, adjusting the posture of the mouse by using hemostatic forceps to enable the abdomen of the mouse to be upward and the whole mouse to be more stretched, transversely placing the mouse, clamping the skin at the lower position of the abdomen of the mouse by using the hemostatic forceps, tearing the skin in the opposite direction by suddenly exerting force, and fully exposing the abdomen.

(ii) The peritoneum is lifted by the sterile ophthalmology bent forceps, then a proper amount of culture medium is injected into the abdominal cavity by a 5mL injector to ensure that the abdomen is fully inflated, the injector is put down, the hemostatic forceps held by two hands are matched to lift the opposite limbs of the mouse up and gently shake, and the macrophage in the abdominal cavity is fully infiltrated and then is sucked out by the same injector and injected into a 1640HT culture medium which is prepared in advance and contains 20% fetal calf serum for later use.

3.2 preparation of thymocytes from feeder cells:

(i) three-week-old BALB/c mice are killed by neck-leading, and soaked in 75% alcohol solution for 5 min; taking out and putting into a sterile plate which is arranged on a superclean workbench in advance, adjusting the posture of the mouse by using hemostatic forceps to enable the abdomen of the mouse to be upward and the whole body to be more stretched, transversely placing the mouse, clamping the skin at the position which is lower than the chest of the mouse by using hemostatic forceps, tearing the skin in the opposite direction by suddenly exerting force, and fully exposing the chest and the abdomen.

(ii) The left hand clamps the sternum tip through the skin of the chest and abdomen with sterile curved forceps, cuts along the outer edge of the sternum body, fully exposes the diaphragm of the thoracic cavity, and always clamps the sternum tip to expose the state of the thoracic cavity as much as possible.

(iii) And (3) cutting a thoracic diaphragm by a right hand-held clean ophthalmologic scissors, fully exposing the thoracic cavity, probing the thoracic cavity by a curved forceps, clamping the root part of the thymus, completely taking out the thymus, putting the thymus into a 200-mesh screen which is placed in a plate and infiltrated by a culture medium for grinding to obtain thymus feeder cell liquid, and transferring the whole thymus feeder cell liquid into the 1640HT culture medium containing 20% fetal calf serum for later use.

3.3 preparation of mouse myeloma cells: 5 days before fusion, 5 bottles of 35cm are needed for recovering mouse myeloma cells for each fusion²Mouse myeloma cells at a density of 90% to 100%.

3.4 preparation of splenocytes:

(i) collecting BALB/c mouse to be fused, collecting blood in orbit, killing the mouse by breaking neck after blood flow stops, soaking in 75% alcohol solution for 5min, and placing on sterile plate in super clean bench, and lying on right side.

(ii) The abdominal cavity was exposed as described in 3.1, the spleen was removed by aseptic surgery by opening the abdominal cavity, ground in a 200 mesh sieve previously placed in a petri dish and infiltrated with culture medium, and a suspension of spleen cells was prepared and transferred to a 50ml sterile centrifuge tube.

(iii) Adding appropriate amount of RPMI-1640 culture solution 30-35ml, removing obvious fat conglomeration and other impurities with elbow, centrifuging at 1500rpm/min for 5 min/time, collecting spleen cells, centrifuging to remove supernatant, adding new culture medium 30-35ml, and repeating the above process for 2 times to clean spleen cells.

(iv) The cells were resuspended in RPMI-1640 medium and counted.

3.5 cell fusion:

(i) 1mL of PEG-1450, 35mL of RPMI-1640 serum-free medium and 200mL of HAT complete medium prepared in advance and containing 20% fetal calf serum were pre-warmed to 37 ℃ before fusion.

(ii) The prepared myeloma cells and spleen cells are mixed according to the proportion of 1 × 10⁸Splenocytes +1 × 10⁷Myeloma cells were mixed at about 10:1 in a 50mL centrifuge tube, centrifuged at 1500 rpm. times.5 min, and after centrifugation, the supernatant was decanted as much as possible and the bottom of the tube was flicked to loosen the cells into a paste.

(iii)1mL of PEG is sucked by a pipette tube of 1mL and added into a centrifugal tube, the mixture is lightly blown and beaten by an elbow tube while being added, the time is controlled to be about 60s, and then 35mL of pre-warmed RPMI-1640 complete culture solution is added to terminate the fusion reaction.

(iv) Standing for 1-5min, centrifuging at 1000rpm × 5min, carefully discarding supernatant, gently flicking with fingers to homogenize cells, adding the above complete culture medium to resuspend cells, transferring into complete culture medium containing feeder layer cells, mixing, spreading in 96-well cell culture plate at 200 ul/well, adding CO, and culturing₂And (5) culturing in an incubator.

(v) After 7 days, the cell supernatants in the wells were subjected to a 100% change with 15% HT complete medium; after 7 days, the supernatant was aspirated for detection.

3.6 selection of hybridomas: differential screening and indirect ELISA screening of the antigen 2 prepared in example 1 are carried out, the antigen is coated by 200ng/mL, each hole is coated by 0.1mL, cell supernatant 50uL is added for detection, and positive cloning holes are selected.

3.7 cloning of hybridoma cells: the limiting dilution method is adopted, cells are firstly diluted in a gradient mode according to a certain concentration, then the cells are inoculated into each hole of a 96-hole cell culture plate, and only one cell in each hole grows as far as possible. The hybridoma monoclonal positive cell strain is cloned repeatedly at least 3 times, and finally, the hybridoma monoclonal positive cell strain is confirmed to be a stable clone strain after 100% of positive is needed.

3.8 antibody typing: adding 50 ul/hole stable cell strain supernatant into a plate coated by the antigen 2 in parallel, placing the plate in an incubator at 37 ℃ for 30min, washing the plate for 5 times, beating the plate to be dry, adding 50 ul/hole IgG1, IgG2a, IgG2b, IgG3 and IgM typing enzyme, reacting for 30min, adding 100 ul/hole developing solution into the washed plate, stopping reading the plate by using a stop solution after 15min, and determining that the antibody is IgG1 type according to the result.

4. Preparation of monoclonal antibody

4.1 production of monoclonal antibody ascites

Sensitized BALB/c mice, after 1 week, resuspension of the stable hybridoma cells in the logarithmic growth phase was centrifuged at 1500rpm for 5min to collect the cells, the precipitated cells were suspended in serum-free medium, and the cell number was adjusted to (1-2). times.10⁶Ascites induction was performed by intraperitoneal injection of 0.5 mL/mouse. And after 7-10 days, collecting ascites after the abdomen of the mouse is obviously enlarged. And centrifuging the collected ascites in a centrifuge tube at 12000rpm for 10min, and collecting a supernatant for later use after centrifugation.

4.2 purification of ascites of monoclonal antibodies

The monoclonal antibody (designated 10D9-1) was obtained in high purity by ammonium sulfate precipitation and Protein A affinity chromatography (available from GE, USA), and the purity of the antibody is identified by 12% SDS-PAGE as shown in FIG. 2.

4.3 Indirect Activity assay of monoclonal antibodies

The EIA method evaluates the reactivity of the purified antibody and antigen, coats antigen 1 and antigen 2 respectively, both coats 200ng/mL, coats 100ul per well, dilutes the antibody to be evaluated to 1ug/mL, 0.1ug/mL, 0.01ug/mL, 0.001ug/mL and 0.0001ug/mL, evaluates the reactivity of the antibody and antigen, and as a result, the antibody and two antigens have good reactivity, which is shown in figure 3.

Example 3 epitope identification of monoclonal antibodies

1. 15 peptide identification

Antigen 1 was diluted with carbonate buffer (20mmol/L CB, pH 9.6) and coated on a pvc plate at 200ng/ml, and the purified antibody was synthesized with DCP1-50aa polypeptide (synthesized, 15aa in polypeptide length, overlapping 5 amino acids one after the other, as shown by underlining in the table, the specific sequence information is shown in table 1) at 100ng/ml antibody: incubating the polypeptide at the same volume of 50ug/ml for 30min to obtain a detection sample; control samples were 100ng/ml antibody: PBS, incubating for 30min, adding the incubated detection sample and control sample into the plate coated with antigen 1 repeatedly in double holes, incubating for 30min, washing the plate for 5 times, adding GAM-HRP (1/5000 for dilution), incubating for 30min, washing the plate for 5 times, adding substrate, and incubating for 15 min. The ELISA reader has a wavelength of 450-: more than 90% shows good competition effect, which indicates that the antibody should recognize the epitope, and the 10D9-1 recognition epitope is located at 31-45aa, and the specific data are shown in the following table 2.

Table 1 DCP15 peptide synthesis information

Serial number	Epitope	Amino acid sequence information
			DP1	1-15	ANTFLEEVRKGNLER
DP2	11-25	GNLERECVEETCSYE
			DP3	21-35	TCSYEEAFEALESST
DP4	31-45	LESSTATDVFWAKYT
			DP5	41-55	WAKYTACETARTPRD

TABLE 210D 9-115 peptide reactivity identification

Sequence numbering	10D9-1
		DP1	-6％
DP2	-4％
		DP3	-4％
DP4	96％
		DP5	-2％

2. 8 peptide identification

Antigen 1 was diluted with carbonic acid buffer (20mmol/L CB, pH 9.6) and coated on a polyvinyl chloride plate at 200ng/ml, and purified 10D9-1 was combined with the synthetic DCP8 peptide (31-45 aa, 8aa in polypeptide length, single amino acid displacement, see Table 3 for specific sequence information) at a ratio of 100ng/ml antibody: incubating the polypeptide at the same volume of 50ug/ml for 30min to obtain a detection sample; control samples were 100ng/ml antibody: PBS, incubating for 30min, adding the incubated detection sample and control sample into the plate coated with antigen 1 repeatedly in double holes, incubating for 30min, washing the plate for 5 times, adding GAM-HRP (1/5000 for dilution), incubating for 30min, washing the plate for 5 times, adding substrate, and incubating for 15 min. The ELISA reader has a wavelength of 450-: more than 90% of the monoclonal antibodies show good competition effect, show that the antibodies should recognize the epitope, and the results show that the monoclonal antibodies against abnormal prothrombin of human and any 8 peptides do not react, and the specific data are shown in the following table 4.

Table 3 DCP8 peptide synthesis information

Sequence numbering	Epitope information	Amino acid sequence information
			DP41	31-38	LESSTATD
DP42	32-39	ESSTATDV
			DP43	33-40	SSTATDVF
DP44	34-41	STATDVFW
			DP45	35-42	TATDVFWA
DP46	36-43	ATDVFWAK
			DP47	37-44	TDVFWAKY
DP48	38-45	DVFWAKYT

TABLE 410D 9-18 peptide reactivity identification

Sequence numbering	10D9-1
		DP41	-2％
DP42	10％
		DP43	33％
DP44	29％
		DP45	-9％
DP46
		4％
DP47			11％
	DP48	10％

Example 4 monoclonal antibody species-specific evaluation

Finding a 31-45aa section on UniProt, wherein the homology of the 31-45aa section and human DCP is higher (more than 70%), particularly, animal serum products of the section are widely applied to the in vitro diagnosis industry, and corresponding sequences of species which can cause cross reaction to generate interference, such as cows, sheep, goats, horses, rabbits and the like, are specifically shown in Table 5, and are classified according to different sites, so that the species of the serum product which is widely applied to the in vitro diagnosis industry are finally synthesized to correspond to 31-45aa polypeptides (font slant, total 8 strips) for evaluation, wherein the evaluation method comprises the following steps: antigen 1 was diluted with carbonate buffer (20mmol/L CB, pH 9.6) and coated on a polyvinyl chloride plate at 200ng/ml, and purified 10D9-1 was mixed with different species of 31-45aa polypeptides synthesized by the manufacturer in a ratio of 100ng/ml antibody: incubating the polypeptide at the same volume of 50ug/ml for 30min to obtain a detection sample; control samples were 100ng/ml antibody: PBS, incubating for 30min, adding the incubated detection sample and control sample into the plate coated with antigen 1 repeatedly in double holes, incubating for 30min, washing the plate for 5 times, adding GAM-HRP (1/5000 for dilution), incubating for 30min, washing the plate for 5 times, adding substrate, and incubating for 15 min. The ELISA reader has a wavelength of 450-: more than 90% of the monoclonal antibodies show good competitive effect, and show that the antibodies should recognize the epitope, and the results show that the monoclonal antibodies against abnormal prothrombin of the invention do not react with 31-45aa of other species, namely, the antibodies specifically recognize 31-45aa of human DCP, and the specific data are shown in the following table 6.

TABLE 5 amino acid sequence information of DCP31-45aa of different species

Numbering	Species (II)	Name of scholars	Epitope information	Amino acid sequence information
					1	Human being	Homosapiens	31-45	LESSTATDVFWAKYT
2	Kiwi fruit	Macacamulatta	31-45	LESLSATDAFWAKYT
					3	Angola warts monkey	Colobusangolensispalliatus	31-45	LESLSATDAFWAKYT
4	Dongfei baboon	Papioanubis	31-45	LESLSATDAFWAKYT
					5	White-top white-eyebrow monkey	cercocebusatys	31-45	LESLSATDAFWAKYT
6	Macaca fascicularis	macacafascicularis	31-45	LESLSATDAFWAKYT
					7	Flat-topped monkey	Macacanemestrina	31-45	LESLSATDAFWAKYT
8	Mouse ear bat	Myotislucifugus	31-45	LESLSATDAFWAKYT
					9	Domestic cattle	Bostaurus	31-45	LESLSATDAFWAKYT
10	Sheep	Ovisaries	31-45	LESPSATDVFWAKYT
					11	White whale	Delphinapterusleucas	31-45	LESPSATDVFWAKYT
12	Chinese river dolphin	Lipotesvexillifer	31-45	LESPSATDVFWAKYT
					13	Dolphin with wide mouth	Tursiopstruncatus	31-45	LESPSATDVFWAKYT
14	Qinling Chuan golden monkey	Rhinopithecusroxellana	31-45	LESPSATDAFWAKYT
					15	Black and white nose painting monkey	Rhinopithecusbieti	31-45	LESPSATDAFWAKYT
16	Sperm whale	Physetermacrocephalus	31-45	LESPSATDAFWAKYT
					17	Domestic horse	Equuscaballus	31-45	LESSSATEAFWAKYT
18	Rabbit	Oryctolaguscuniculus	31-45	LESSSATEAFWAKYT
					19	Home mouse	Musmusculus	31-45	LESPQDTDVFWAKYT
20	White rat	Rattusnorvegicus	31-45	LESPQDTDVFWAKYT
					21	Goat	Caprahircus	31-45	LESPGATDVFWAKYT
22	Chicken of jungle fowl	Gallusgallus	31-45	LESTVDTDAFWAKYT

TABLE 6 Cross-reactivity of 10D9-1 of the present invention to different species 31-45aa

Example 5 evaluation of the effects of the antibody enzyme immunoassay platform

DCP antibody 11H8-3 (catalog of Enbobmi products, cat # M3404, conjugated DCP 11-25aa amino acid sequence), 5ug/ml coated with 20mmol/L PB7.4 in buffer solution, 100 ul/well at 37 ℃ for 2 hours on polyvinyl chloride plates, labeled with horseradish peroxidase at 10D9-1 concentration of 1 mg/ml. The DCP quality control, the sample serum, the positive control and the blank control are respectively added into corresponding holes for 30ul, the holes are placed at 37 ℃ for incubation for 30min, the washing plate is added with 10D9-1-HRP (20ug/ml) for incubation for 30min for 5 times, the washing plate is added for 5 times, and the substrate is added for incubation for 15 min. The ELIASA has a reading value of 450-620, and the measurement result is calculated back according to the standard curve, which shows that the antibody pairing detection has better correlation with the background (Yapei), and is specifically shown in Table 7.

TABLE 7 evaluation results of antibody enzyme immunization platform

Background value (mAU/ml)	Constant value
		11364	10655
15085	14144
		2151	2017
3756	3522
		8873	8319
4961	4651
		1010	947
455	427
		683	640

Example 6 evaluation results of antibody luminescence plateau

6.111H 8-3 antibody magnetic particle coating

(1) And (3) balancing the magnetic particles and the EDC to room temperature, taking the magnetic particles with the concentration of 4mg/ml, adding a certain concentration of activating agent EDC, and standing at room temperature for activation for 30min.

(2) And taking the activated magnetic particles, removing supernatant after magnetic collection by using magnetic collection equipment, and adding MES buffer solution for resuspension.

(3) Adding a certain amount of 11H8-3 antibody, mixing uniformly, and reacting at room temperature for 3H.

(4) And (3) removing supernatant after magnetic collection by using a magnetic frame, adding phosphate buffer solution containing certain protein with the same volume, and reacting for 3 hours at room temperature.

(5) And (4) removing supernatant after magnetism is collected by a magnetism collecting device, and adding an equal volume of cleaning solution for repeated washing for 4 times.

(6) And (4) storing the washed magnetic particles in a magnetic bead diluent, and storing at 2-8 ℃.

6.2 the invention of the monoclonal antibody acridinium ester marker against abnormal prothrombin

(1) And adding acridinium ester into the anti-human abnormal prothrombin monoclonal antibody containing phosphate buffer solution, uniformly mixing, and reacting for 30min at room temperature in a dark place.

(2) Adding termination buffer solution, mixing uniformly, and reacting for 30min at room temperature in a dark place.

(3) After the labeled acridinium ester is dialyzed, the same volume of glycerol is added firstly and mixed evenly, then 10% BSA is added and mixed, and the mixture is stored in the dark at the temperature of minus 20 ℃.

6.311H8-3-10D9-1 DCP prototype reagent on-machine detection

And (3) placing the constructed DCP reagent on a full-automatic chemiluminescence instrument Caris200, and detecting the sample according to the standard operation protocol of the full-automatic chemiluminescence instrument Caris 200. A commercial reagent Yapek DCP chemiluminescence detection reagent which is the mainstream in the market is selected as a contrast reagent, 58 clinical samples are detected in parallel by the constructed DCP prototype detection reagent and the commercial DCP detection reagent (Yapek) strictly according to the operation of a commercial reagent specification, the correlation coefficient r value of the detection results of the two reagents is 0.981, and the prototype reagent which is constructed by using an antibody 10D9-1 for specifically identifying human DCP has an excellent detection effect, and is shown in figure 4.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Sequence listing

<110> Xiamen Yingbo Mi Biotech Co., Ltd

<120> anti-human abnormal prothrombin antibody and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Human Des-γ-carboxyprothrombin VH CDR1

<400> 1

Gly Tyr Thr Phe Thr Ser Tyr Val

1 5

<210> 2

<211> 8

<212> PRT

<213> Human Des-γ-carboxyprothrombin VH CDR2

<400> 2

Ile Phe Pro Tyr Asn Asp Val Thr

1 5

<210> 3

<211> 8

<212> PRT

<213> Human Des-γ-carboxyprothrombin VH CDR3

<400> 3

Ala Arg Trp Gly Gly Arg Asn Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> Human Des-γ-carboxyprothrombin VL CDR1

<400> 4

Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr

1 5 10

<210> 5

<211> 3

<212> PRT

<213> Human Des-γ-carboxyprothrombin VL CDR2

<400> 5

Leu Val Ser

1

<210> 6

<211> 9

<212> PRT

<213> Human Des-γ-carboxyprothrombin VL CDR3

<400> 6

Trp Gln Gly Thr His Phe Pro Tyr Thr

1 5

<210> 7

<211> 113

<212> PRT

<213> Human Des-γ-carboxyprothrombin VH

<400> 7

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

Gly Tyr Ile Phe Pro Tyr Asn Asp Val Thr Glu Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Trp Gly Gly Arg Asn Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val

<210> 8

<211> 113

<212> PRT

<213> Human Des-γ-carboxyprothrombin VL

<400> 8

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

1 5 10 15

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

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 10

<211> 1869

<212> DNA

<213> Human prothrombin

<400> 10

atggcgcacg tccgaggctt gcagctgcct ggctgcctgg ccctggctgc cctgtgtagc 60

cttgtgcaca gccagcatgt gttcctggct cctcagcaag cacggtcgct gctccagcgg 120

gtccggcgag ccaacacctt cttggaggag gtgcgcaagg gcaacctgga gcgagagtgc 180

gtggaggaga cgtgcagcta cgaggaggcc ttcgaggctc tggagtcctc cacggctacg 240

gatgtgttct gggccaagta cacagcttgt gagacagcga ggacgcctcg agataagctt 300

gctgcatgtc tggaaggtaa ctgtgctgag ggtctgggta cgaactaccg agggcatgtg 360

aacatcaccc ggtcaggcat tgagtgccag ctatggagga gtcgctaccc acataagcct 420

gaaatcaact ccactaccca tcctggggcc gacctacagg agaatttctg ccgcaacccc 480

gacagcagca ccacgggacc ctggtgctac actacagacc ccaccgtgag gaggcaggaa 540

tgcagcatcc ctgtctgtgg ccaggatcaa gtcactgtag cgatgactcc acgctccgaa 600

ggctccagtg tgaatctgtc acctccattg gagcagtgtg tccctgatcg ggggcagcag 660

taccaggggc gcctggcggt gaccacacat gggctcccct gcctggcctg ggccagcgca 720

caggccaagg ccctgagcaa gcaccaggac ttcaactcag ctgtgcagct ggtggagaac 780

ttctgccgca acccagacgg ggatgaggag ggcgcgtggt gctatgtggc cgggaagcct 840

ggcgactttg ggtactgcga cctcaactat tgtgaggagg ccgtggagga ggagacagga 900

gatgggctgg atgaggactc agacagggcc atcgaagggc gtaccgccac cagtgagtac 960

cagactttct tcaatccgag gacctttggc tcgggagagg cagactgtgg gctgcgacct 1020

ctgttcgaga agaagtcgct ggaggacaaa accgaaagag agctcctgga atcctacatc 1080

gacgggcgca ttgtggaggg ctcggatgca gagatcggca tgtcaccttg gcaggtgatg 1140

cttttccgga agagtcccca ggagctgctg tgtggggcca gcctcatcag tgaccgctgg 1200

gtcctcaccg ccgcccactg cctcctgtac ccgccctggg acaagaactt caccgagaat 1260

gaccttctgg tgcgcattgg caagcactcc cgcaccaggt acgagcgaaa cattgaaaag 1320

atatccatgt tggaaaagat ctacatccac cccaggtaca actggcggga gaacctggac 1380

cgggacattg ccctgatgaa gctgaagaag cctgttgcct tcagtgacta cattcaccct 1440

gtgtgtctgc ccgacaggga gacggcagcc agcttgctcc aggctggata caaggggcgg 1500

gtgacaggct ggggcaacct gaaggagacg tggacagcca acgttggtaa ggggcagccc 1560

agtgtcctgc aggtggtgaa cctgcccatt gtggagcggc cggtctgcaa ggactccacc 1620

cggatccgca tcactgacaa catgttctgt gctggttaca agcctgatga agggaaacga 1680

ggggatgcct gtgaaggtga cagtggggga ccctttgtca tgaagagccc ctttaacaac 1740

cgctggtatc aaatgggcat cgtctcatgg ggtgaaggct gtgaccggga tgggaaatat 1800

ggcttctaca cacatgtgtt ccgcctgaag aagtggatac agaaggtcat tgatcagttt 1860

ggagagtag 1869

<210> 10

<211> 150

<212> DNA

<213> Human Des-γ-carboxyprothrombin

<400> 10

atggcgcacg tccgaggctt gcagctgcct ggctgcctgg ccctggctgc cctgtgtagc 60

cttgtgcaca gccagcatgt gttcctggct cctcagcaag cacggtcgct gctccagcgg 120

gtccggcgag ccaacacctt cttggaggag 150

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agatctatgg cgcacgtccg aggcttgc 28

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gaattcctcc tccaagaagg tgttggctcg cc 32

<210> 13

<211> 50

<212> PRT

<213> Human Des-γ-carboxyprothrombin

<400> 13

Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu

35 40 45

Glu Glu

50

Claims (10)

1. The anti-human abnormal prothrombin antibody is characterized by comprising a heavy chain variable region and a light chain variable region, and can be specifically combined with human abnormal prothrombin, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6.

2. The antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 7 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 8.

3. The anti-human abnormal prothrombin antibody according to any one of claims 1 or 2, wherein the antibody is one or more of a monoclonal antibody, a single chain antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody or a Fab fragment.

4. A nucleic acid encoding the anti-human abnormal prothrombin antibody according to any one of claims 1 or 2.

5. A vector comprising the nucleic acid of claim 4.

6. A transgenic cell line comprising the nucleic acid of claim 4.

7. A recombinant bacterium comprising the nucleic acid according to claim 4.

8. A recombinant virus comprising the nucleic acid of claim 4.

9. Use of the anti-human abnormal prothrombin antibody according to any one of claims 1 to 3 in the preparation of a hepatocellular carcinoma detection kit.

10. A kit comprising the antibody of any one of claims 1 to 3.

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