CN108659131B

CN108659131B - anti-CEACAM-5 single-domain antibody and application thereof

Info

Publication number: CN108659131B
Application number: CN201810525042.5A
Authority: CN
Inventors: 李文秋; 谢英林; 徐雪松; 王珂; 刘永红
Original assignee: Changchun Lite Biotechnique Co ltd
Current assignee: Changchun Lite Biotechnique Co ltd
Priority date: 2018-05-28
Filing date: 2018-05-28
Publication date: 2021-09-14
Anticipated expiration: 2038-05-28
Also published as: CN108659131A

Abstract

The invention discloses a single domain antibody of anti-CEACAM-5, the single domain antibody has 3 unique complementarity determining regions CDR1, CDR2 and CDR3, the invention also provides an expression vector containing the variable region coding sequence of the single domain antibody, a host cell containing the expression vector, the invention also provides a fusion protein of the variable region of the single domain antibody and human alkaline phosphatase, the application of the single domain antibody in the preparation of a CEACAM-5 detection kit, a method for carrying out CEACAM-5 immunodetection by using the single domain antibody, and a corresponding detection kit. The anti-CEACAM-5 single-domain antibody provided by the invention has specific recognition and binding capacity to CEACAM-5, the single-domain antibody affinity can reach 4.51E-09, and the antibody has a unique antigenic determinant recognition site, and can obtain excellent detection effect in CEACAM-5 immunoassay, particularly in a double-antibody sandwich method.

Description

anti-CEACAM-5 single-domain antibody and application thereof

Technical Field

The invention discloses a polypeptide, and more particularly discloses an immunoglobulin.

Background

Carcinoembryonic antigen (CEACAM-5, abbreviated CEA, also known as CD66e) is a glycoprotein having a molecular weight of about 180 kDa. CEACAM-5 is a member of the immunoglobulin superfamily and contains 7 domains linked to the cell membrane via a Glycosylphosphatidylinositol (GPI) anchor. The 7 domains include a single N-terminal Ig variable domain and 6 domains homologous to Ig constant domains (a1-B1-a 2-B2-A3-B3). CEACAM-5 was originally classified as a protein expressed only in fetal tissues and has now been identified in several normal adult tissues. Overexpression of CEACAM-5 is observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Thus, CEACAM-5 has been identified as a tumor associated antigen. CEACAM-5 is readily cleaved from the cell surface and shed from the tumor into the bloodstream, either directly or via the lymphatic system. Because of this property, the level of serum CEACAM-5 has been used as a clinical marker to diagnose and screen for cancer. Furthermore, CEACAM-5 has also been used as a tumor marker, and immunological assays for measuring increased CEACAM-5 in the blood of cancer patients have been used clinically for prognosis and control of cancer.

More importantly, CEACAM-5 has become a potentially useful tumor-associated antigen for targeted therapy. There have been reported 2 main approaches to cancer targeted immunotherapy using CEACAM-5. A method for inducing the lytic activity of immune cells, in particular by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), using an anti-CEACAM-5 antibody, in order to eliminate CEACAM-5 expressing tumor cells; another approach is to specifically target CEACAM-5 expressing tumor cells by conjugating anti-CEACAM-5 antibodies or antibody fragments to effector molecules such as drugs, toxins, radionucleotides, immunomodulators or cytokines, thereby exerting the therapeutic effect of the effector molecules.

Various monoclonal antibodies have been generated against CEACAM-5. Chester et al have isolated single chain anti-CEACAM-5 antibodies from phage display libraries for use in radioimmunoassay and radioimmunotherapy (U.S. Pat. No.5,876,691), followed by humanization of the antibodies (U.S. Pat. No.7,232,888). Radiolabeled anti-CEACAM-5 antibodies have been used in clinical trials in patients with colorectal cancer.

In 1993, Hamers-Casterman et al found that a class of heavy chain only dimeric antibodies H was found in camelids (camels, dromedary and llamas) in vivo₂It is mainly of the IgG2 and IgG3 type. Such antibodies are lacking a light chain, and thus are referred to as heavy chain-only antibodies (HCabs), and their antigen-binding portionsA site consists of one domain, called VHH region, and therefore this class of antibodies is also called single domain antibodies or single domain antibodies (sdabs). Since this type of antibody is a variable region sequence after removal of a constant region, the molecular weight is only 15kD, and the diameter is about 10 nm, and thus it is also called nanobody (Nbs). In addition, such single domain antibodies, called VNARs, are also observed in sharks. This heavy chain-only antibody, originally recognized only as a pathological form of human B-cell proliferative disease (heavy chain disease), may be due to mutations and deletions at the genomic level resulting in the inability of the heavy chain CH1 domain to be expressed, such that the expressed heavy chain lacks CH1 and thus lacks the ability to bind to the light chain, thereby forming a heavy chain dimer.

Single domain antibodies are comparable in affinity to their corresponding scFv, but surpass scfvs in solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination, relative to scfvs of conventional four-chain antibodies.

Single domain antibodies have minimal functional antigen binding fragments derived from HCAbs in adult camelids, have high stability and high avidity for binding to antigen, and interact with protein cleft and the active site of the enzyme, making it act like inhibitors. Thus, single domain antibodies may provide a new concept for designing small molecule enzyme inhibitors from peptidomimetic drugs. Due to the heavy chain only, single domain antibodies are easier to manufacture than mabs. The unique properties of single domain antibodies, such as stability in extreme temperature and pH environments, can be made in large yields at low cost. Therefore, the single-domain antibody has great value in the treatment and diagnosis of diseases, and has great development prospect in the antibody target diagnosis and treatment of tumors.

In view of the fact that CEACAM-5 is more overexpressed in some solid tumors such as colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatoma, breast cancer and thyroid cancer, the development of single-domain antibodies against CEACAM-5 has made full use of the super-strong antigen recognition ability of single-domain antibodies, and particularly, the recognition of some antigenic determinants hidden in fissures or cavities is a new need in the field of antibody technology. However, the existence of some structural defects such as low affinity, easy aggregation, short serum half-life, etc., due to the low molecular weight of the single domain antibody, has prevented the further application of the single domain antibody. In the specific application of CEACAM-5 in immunoassay, if the anti-CEACAM-5 antibody recognizes the epitope of CEACAM-5, which is too single or too close or overlapping in sites, the specific antigen-antibody binding reaction is infected, thereby seriously affecting the detection efficiency. The invention aims to provide a single-domain antibody which can fully exert the excellent performance of the single-domain antibody and overcome the inherent defects of the single-domain antibody, namely, the antibody has high specific antigen recognition capability, high affinity, unique epitope recognition sites and excellent detection efficiency in the immunoassay of CEACAM-5 antigen, particularly in a double-antibody sandwich method.

Disclosure of Invention

Based on the above objects, the present invention provides a single domain antibody against CEACAM-5, wherein the variable region of the single domain antibody has 3 complementarity determining regions CDR1, CDR2, and CDR3, wherein the CDR1 sequence consists of the amino acid sequence depicted in SEQ ID No.1, the CDR2 sequence consists of the amino acid sequence depicted in SEQ ID No.2, and the CDR3 sequence consists of the amino acid sequence depicted in SEQ ID No. 3. The antibodies have unique epitope recognition sites.

In a preferred embodiment, the variable region sequence of the single domain antibody consists of the amino acid sequence of SEQ ID NO. 4. A preferred embodiment of a single domain antibody having this variable region sequence in the present invention is single domain antibody 2E 5.

Secondly, the invention provides the fusion protein of the single domain antibody and the human alkaline phosphatase, the fusion protein is formed by connecting the single domain antibody or the variable region fragment thereof and a human placental alkaline phosphatase reporter gene protein in series, and in a preferred embodiment of the invention, the sequence of the human placental alkaline phosphatase reporter gene protein is shown as SEQ ID NO. 8.

Thirdly, the invention also provides a nucleotide coding sequence of the single domain antibody sequence, and the coding sequence is shown by SEQ ID NO. 5.

Fourthly, the invention provides an expression vector containing the nucleotide coding sequence, and the vector is pMES 4.

Fifth, the present invention provides a host cell containing the above expression vector, wherein the cell is Escherichia coli BL₂₁(DE₃)。

Sixth, the invention also provides application of the single domain antibody in preparation of a CEACAM-5 antigen detection reagent.

Seventhly, the invention provides a CEACAM-5 antigen immunodetection method based on non-diagnostic purposes, the method is a double-antibody sandwich enzyme-linked immunoassay, the variable region sequence of the primary antibody contains an amino acid sequence shown in SEQ ID NO.6, the secondary antibody is an enzyme-linked secondary antibody, and the variable region sequence of the secondary antibody contains an amino acid sequence shown in SEQ ID NO. 4.

In a preferred technical scheme, the enzyme-linked secondary antibody is a fusion protein of a single-domain antibody of anti-CEACAM-5 antigen and human placental alkaline phosphatase, and in a preferred embodiment of the invention, the sequence of the human placental alkaline phosphatase is shown as SEQ ID NO. 8.

Finally, the invention also provides an immunoassay kit for detecting CEACAM-5 antigen by using a double-antibody sandwich method, the kit comprises a primary antibody for capturing the antigen and a secondary antibody for combining with the antigen to initiate an enzyme-linked reaction, the variable region sequence of the primary antibody contains the amino acid sequence shown in SEQ ID NO.6, the secondary antibody is a fusion protein of a single-domain antibody of the anti-CEACAM-5 antigen and human alkaline phosphatase, and the fusion protein sequence contains the amino acid sequence shown in SEQ ID NO. 7.

The single-domain antibody 2E5 for resisting the CEACAM-5 antigen has specific recognition and binding capacity on the CEACAM-5 antigen, the affinity of the single-domain antibody can reach 4.51E-09, and compared with a plurality of anti-CEACAM-5 single-domain antibodies, the single-domain antibody can recognize different antigenic determinants and has a unique antigenic determinant recognition site. The fusion protein of the single domain antibody of the CEACAM-5 antigen and the human alkaline phosphatase provided by the invention not only retains the specific recognition and knot of the CEACAM-5 antigenThe antibody has the advantages of high binding capacity, high complete activity of human alkaline phosphatase, good application prospect in the application of detecting the CEACAM-5 antigen content in a specimen by a double-antibody sandwich immunoassay method, particularly unique antigen recognition sites, capability of being matched with a plurality of other anti-CEACAM-5 antibodies to be used as a primary antibody and a secondary antibody respectively, greatly improved detection efficiency, ensured detection specificity, particularly good linear curve of the antibody in the matching use with a 2C2 single-domain antibody, and R²0.9934 is reached, approaching a completely linear relationship.

Drawings

FIG. 1 is an SDS-PAGE identification of single domain antibody 2E 5;

FIG. 2-graph of the single domain antibody 2E5 affinity test;

FIG. 3 is a graph of the results of a cross-reactivity assay for single domain antibody 2E 5;

figure 4.Biacore analysis single domain antibody 2E5 binding site flow chart.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 construction and screening of Single Domain antibody phage display library against CEACAM-5

1.1 immunization of alpaca: selecting one healthy adult alpaca, uniformly mixing a recombinant protein CEACAM-5 antigen and Freund's adjuvant according to a ratio of 1:1, immunizing the alpaca by adopting a back subcutaneous multipoint injection mode according to 6-7 mu g/Kg for four times, wherein the immunization interval is 2 weeks. And collecting alpaca peripheral blood for constructing a phage display library.

1.2 separation of camel source lymphocytes: lymphocytes were analyzed from collected camel-derived anticoagulated whole blood according to routine procedures in the art, every 2.5X 10⁷1mL of RNA isolation reagent was added to each living cell, 1mL of the reagent was extracted with RNA, and the remaining cells were stored at-80 ℃.

1.3 Total RNA extraction: total RNA was extracted according to the routine procedures in the art and adjusted to 1. mu.g/. mu.L with RNase-free water.

1.4 reverse transcription Synthesis of cDNA:

reverse transcription of cDNA was carried out using RNA obtained in 1.3 steps as a template according to the instructions of a reverse transcription KIT (Transcriptor first stand cDNA Synthesis KIT from Roche Co.)

1.5 antibody variable region Gene amplification: and carrying out PCR reaction by using cDNA obtained by reverse transcription as a template. Amplification was performed in two rounds, and the primer sequences for the first round of PCR were as follows:

CALL001：GTCCTGGCTGCTCTTCTACAAGG

CALL002：GGTACGTGCTGTTGAACTGTTCC

the PCR reaction conditions and procedures were: 5min at 95 ℃; 30cycles at 95 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 30 s; 7min at 72 DEG C

The agarose gel recovery kit gel was used to recover a band of about 700bp, and the nucleic acid concentration was finally adjusted to 5 ng/. mu.l with water.

The primer sequences for the second round of PCR were as follows:

VHH-Back：GATGTGCAGCTGCAGGAGTCTGGRGGAGG

VHH-For：CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

the PCR reaction conditions and procedures were: 5min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 15 cycles; 7min at 72 DEG C

PCR products were purified using a PCR product recovery kit.

1.6 vector construction

Carrying out PstI and BstEII double enzyme digestion on pMES4 and the second PCR product respectively, taking 1.5 mu g of the vector after enzyme digestion and 450ng of the second PCR product after enzyme digestion, adding 15 mu L T4DNA ligase, supplementing buffer solution and water to the total volume of 150 mu L, carrying out overnight ligation at 16 ℃, and recovering the ligation product. Product recovery was performed using a PCR product recovery kit, eluting with 20. mu.L water.

1.7 electrotransformation and determination of the storage volume

mu.L of the purified ligation product was taken, added to a pre-cooled electric cuvette containing 50. mu.L of E.coli TG1 competent cells, and placed in an electric converter (ECM 630 electric converter of BTX, USA) for electric conversion, and the electric cuvette was taken out, and the transformant was recovered and cultured. Randomly selecting clones, and carrying out colony PCR identification. The pool capacity (pool capacity ═ number of clones × dilution × [ positive rate ] PCR assay × 10) was estimated from the PCR positive rate.

The primer sequences are as follows:

MP57：TTATGCTTCCGGCTCGTATG

GIII：CCACAGACAGCCCTCATAG

1.8 phage amplification

Inoculating recovered bacteria solution into YT-AG culture medium, culturing at 37 deg.C and 200rpm until culture OD₆₀₀0.5. 10ml of the bacterial suspension was taken out and added to 4X 10¹⁰VCSM13, 30min at 37 ℃ for static infection. Centrifuging at 4000rpm at normal temperature for 10min, and removing supernatant. The cells were resuspended in 2 XYT-AK (ampicillin and kanamycin-containing) medium and cultured overnight at 37 ℃ and 200 rpm. Centrifuging, taking a supernatant in a 40ml tube, adding 10ml of PEG/NaCl (20%/2.5M) solution, mixing thoroughly, centrifuging, discarding the supernatant, washing the precipitate with 1ml of ice PBS, centrifuging, taking 250 μ l of precooled PEG/NaCl from the supernatant, mixing thoroughly, washing and resuspending.

Determining the phage titer: TG1 was cultured to OD₆₀₀When the phage was diluted with LB medium in a gradient manner at 0.4, the phage TG1 culture was mixed and cultured in a double dilution manner, and the plaque formation in the plate was observed the next day, and the number of plaques was counted on a dilution gradient plate of 30 to 300 and the phage titer (pfu) was calculated according to the following equation.

Phage titer (pfu/ml) dilution times plaque number times 100

1.9 Single Domain antibody screening

Positive clones were screened by ELISA with recombinant CEACAM-5 antigen. ELISA plates were coated with recombinant CEACAM-5 antigen, blocked with 5% BSA, and washed with PBST. Mu.l phage supernatant was added to each well and left at 37 ℃ for 1 h. The supernatant was discarded, and a secondary HRP-labeled mouse anti-M13 antibody was added thereto and the mixture was left at 37 ℃ for 1 hour. Discarding the supernatant, adding TMB solution, incubating at room temperature for 5min, adding 2M sulfuric acid stop solution into each well, and reading with microplate reader at 450 nm.

And selecting the clone with positive phage ELSIA result, and extracting the plasmid. The sequencing results were analyzed using Vector NTI software, and the entries IMGT (see Table II)https://www.imgt.org/IMGT_vquest) Antibody light and heavy chain genes were analyzed to determine the framework regions (framework regions) of the variable regionss, FR) and Complementarity Determining Regions (CDRs).

The nucleotide sequence of the heavy chain of the anti-CEACAM-5 antigen single domain antibody 2E5 is shown as SEQ ID NO.5, and the amino acid sequence of the variable region is shown as SEQ ID NO.4, wherein the amino acid sequence at the 1st to 25 th positions is FR1, the amino acid sequence at the 26 th to 32 th positions is CDR1, the amino acid sequence at the 33 th to 48 th positions is FR2, the amino acid sequence at the 49 th to 56 th positions is CDR2, the amino acid sequence at the 57 th to 94 th positions is FR3, the amino acid sequence at the 95 th to 105 th positions is CDR3, and the amino acid sequence at the 106 th position 116 is FR 4.

EXAMPLE 2 preparation of anti-CEACAM-5 antigen Single Domain antibody 2E5

2.1 amplification of original strain TG1 of single-domain antibody and transformation of E.coli BL21(DE3) with recombinant plasmid of single-domain antibody

The original strain TG1 glycerol containing single domain antibody nucleic acid was inoculated according to 1: the culture was inoculated at 1000 ratio to 5mL of fresh LB-A medium and cultured overnight at 37 ℃ and 200 rpm. The following day, Plasmid was extracted using a Plasmid mini kit (OMEGA) as per the instructions. After verification, 1. mu.l of the plasmid is transformed into 100. mu.l of competent cells, gently mixed, placed on ice for 30min, hot shocked in a water bath at 42 ℃ for 90s, and cooled in an ice bath for 3 min. 600. mu.l of LB medium was added to the centrifuge tube, and the mixture was cultured with shaking at 37 ℃ for 60 min. 100. mu.l of the supernatant was applied to an LB-A plate using a triangle spreader and cultured overnight at 37 ℃ in an inverted state.

2.2 inducible expression of Single Domain antibodies

The above monoclonal colonies were picked up in LB-A medium and cultured overnight with shaking at 37 ℃. The next day, the bacterial liquid was taken according to the ratio of 1: adding 100ml of fresh LB-A culture medium in a proportion of 100, and performing shaking culture at 37 ℃ for 3h until the bacterial liquid OD₆₀₀After adding IPTG to a final concentration of 1mM, the mixture was induced overnight at 30 ℃. On the third day, 8000rpm, centrifugation for 10min collected the thalli, and 1.5mL of precooled TES buffer was added to resuspend the pellet. After 2min in ice bath, gently shake for 30s and repeat the cycle 6 times. 3.0ml TES/4 (TES diluted 4 times with water) was added, gently shaken for 30s, and then allowed to stand on an ice bath for 2min, and the shaking and standing steps were repeated a total of 6 times. Centrifuging at 9000rpm and 4 deg.C for 10min, and collecting supernatant as periplasm extract.

2.3 purification and characterization of Single Domain antibodies

After resuspending IMAC Sepharose (GE Co., Ltd.), 2ml was taken and added to a gravity column, and the mixture was allowed to stand for 30min to allow Sepharose to naturally settle at the bottom of the gravity column, and the preservation buffer was discharged. Adding 2 times of column volume of nickel sulfate solution (0.1M), and flowing out the nickel sulfate solution at the flow rate of about 8 s/drop; adding 10 times of column volume of balance buffer solution to balance and wash sepharose, and keeping the flow rate unchanged; diluting the sample by 2 times of a balance buffer solution, adding the diluted sample into a gravity column, adjusting the flow rate to be 6 s/drop, and collecting the penetration liquid; adding 10 times of column volume of washing buffer solution to wash sepharose, maintaining the flow rate unchanged, and collecting washing solution; adding elution buffer solution with the volume 3 times of that of the column, maintaining the flow rate at 6 s/drop, and collecting the eluent containing the target protein; finally, the sepharose is washed by sequentially adding 10 times of column volume of the equilibrium buffer, 10 times of column volume of pure water and 10 times of column volume of 20% ethanol, and finally 4ml of 20% ethanol is reserved for storing the chromatographic column. The samples collected above were subjected to SDS-PAGE (FIG. 1: M is a rainbow 180 broad-spectrum protein Marker; 1 is a purified single-domain antibody 2E 5).

Example 3 affinity Activity of anti-CEACAM-5 Single Domain antibodies with CEACAM-5 antigen

3.1 chip antigen coupling

The CEACAM-5 antigen is prepared into 20 mu g/mL working solution by using sodium acetate buffer solutions with different pH values (pH 5.5, pH 5.0, pH 4.5 and pH 4.0), meanwhile, 50mM NaOH regeneration solution is prepared, the electrostatic binding between the antigen and the surface of a chip (GE company) under different pH conditions is analyzed by utilizing a template method in a Biacore T100 protein interaction analysis system instrument, a proper most neutral pH system is selected by taking the quantity of signal increase to reach 5 times RL as a standard, and the antigen concentration is adjusted as a condition during coupling according to requirements. Coupling the chip according to a template method carried by the instrument: wherein, the 1 channel selects a blank coupling mode, the 2 channel selects a Target coupling mode, and the Target is set as a designed theoretical coupling quantity. The coupling procedure took approximately 60 min.

3.2 analyte concentration setting Condition exploration and regeneration Condition optimization

A manual sample injection mode is adopted, a1, 2-channel 2-1 mode is selected for sample injection, and the flow rate is set to be 30 mu L/min. The sample injection conditions are 120s and 30 mu L/min. Regeneration conditions were all 30s, 30. mu.L/min. The buffer was run continuously empty first until all baselines were stable. Single domain antibody solutions with larger concentration spans were prepared in running buffer formulations, suggesting settings of 200. mu.g/mL, 150. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 2. mu.g/mL. Preparing a regeneration solution, selecting the regeneration solution with four pH gradients of a glutamate acid system: 1.5,2.0,2.5,3.0. A200. mu.g/mL sample of analyte was manually injected and the 2-channel was observed, regenerating from the most neutral pH regeneration buffer until the line of response after 2-channel regeneration returned to the same height as the baseline. And manually injecting a sample of 200 mu g/mL of analyte once again, observing the signal change of the 2-1 channel and recording the binding capacity, regenerating by using a regeneration solution which finally returns the response line to the base line in the previous step, then manually injecting a sample of 200 mu g/mL of analyte once again, observing the signal change of the 2-1 channel and recording the binding capacity, comparing with the value of the previous binding capacity, if the deviation is less than 5 percent, determining that the regeneration solution with the pH value is the optimal regeneration solution, and if the binding capacity of re-injection is lower, continuing to perform the experiment by using a regeneration buffer solution with lower pH value. And taking the selected optimal regeneration solution as a chip surface regeneration reagent after each sample introduction. And respectively injecting analyte concentration samples arranged on the sample injection device, and analyzing the binding capacity of each concentration to finally determine the concentration gradient required by the affinity test.

3.3 affinity assay

According to the optimized sample concentration gradient, the solution is regenerated, and the affinity between the single-domain antibody and the antigen is tested by using a template method carried by the instrument (wherein the sample injection condition is set to be 60s and 30 mu L/min; the dissociation time is 600s, and the regeneration condition is set to be 30s and 30 mu L/min). The signal condition of the 2-1 channel is observed at any time. The affinity testing process takes approximately 200 min.

3.4 analysis of results

The binding dissociation curves of a plurality of appropriate concentration gradients are selected and all curves are fitted by adopting a 1:1binding mode, and important parameters such as an affinity value, a binding constant and a dissociation constant are finally obtained (figure 2: the binding constant of the single-domain antibody 2E5 and the CEACAM-5 antigen is 2.186E +5, and the dissociation constant is 9.864E-5). The affinity value of the anti-CEACAM-5 single-domain antibody 2E5 is 4.51E-9

Example 4 Cross-reactivity assay for anti-CEACAM-5 Single Domain antibody 2E5

Coating the antigens CEACAM5, CEACAM1 and CEACAM6 with 0.2 μ g/well of an enzyme label plate respectively, and incubating overnight at 4 ℃; the next day, 200ul blocking solution (1% BSA) was added to each well, and after incubation for 1h at 37 ℃, PBST was washed 5 times for 3min each; adding 50 μ l of 3 μ g single domain antibody 2E5 into each well, incubating at 37 deg.C for 0.5h, and washing with PBST for 5 times, each for 3 min; using goat anti-alpaca antibody (GE company) marked by HRP as a secondary antibody, and incubating for 0.5h at 37 ℃ according to the dilution ratio of the gradient and 50 ul/hole; adding TMB chromogenic substrate, reacting at room temperature in dark for 10min, adding 2M H₂SO₄The reaction was stopped and the OD read at 450 nm. Negative alpaca serum was also used as a control. The results are shown in fig. 3, where the working concentration of the two antigens CEACAM1 and CEACAM6 reached 1: at 2000, the detected OD value is still lower than that of negative serum, which indicates that the specificity of the single-domain antibody of the item is good.

Example 5 ELISA overlay data analysis of Single Domain antibody 2E5

4.1 determination of the saturation concentration of antigen

CEACAM-5 antigen was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated at 4 ℃ for 24h, and washed 5 times. Blocking was performed overnight with 1% BSA as blocking agent and the plate was washed 5 times. Single-domain antibodies diluted in different gradients, a negative control (negative serum 1:100), a PBS blank control, incubation at 37 ℃ for 30min, and washing for 5 times are added into the ELISA plate. Adding 1: the goat anti-alpaca IgG labeled with HRP diluted at the ratio of 4000 was incubated at 37 ℃ for 30min, and the plate was washed 5 times. Adding TMB developing solution, incubating at 37 ℃ for 10min, and stopping reaction by 2M sulfuric acid. Reading the light absorption value of 450nm, drawing an antibody saturation curve, and selecting the concentration which does not increase with the increase of the concentration as the saturation concentration according to the result.

4.2 site overlay experiments

The first antibody is added for reaction, the second antibody is added after the plate is washed, the enzyme-labeled secondary antibody is added after the plate is washed, and the color reading of TMB is carried out (the method is the same as 4.1). And calculating the overlapping rate AI of the two antibodies, wherein the AI is more than 50 percent, which indicates that the antigenic sites of the 2 antibodies to be detected are different, the AI is less than 50 percent, which indicates that the antigenic epitopes of the two antibodies to be detected are the same, and the larger the AI value is, the lower the possibility of site overlapping is. The formula is as follows: AI [2 a (1+2) - (a1+ a2) ]/a (1+2) × 100%

A1-first Strain antibody reading

A2-second Strain antibody reading

A (1+2) -overlay of 2 antibody readings

Table 1: antibody epitope superposition experiment

	1st antibody	2nd antibody	1st antibody +2nd antibody	Overlap ratio
					2E5+11C12	0.335	0.529	0.762	86.61％
2E5+15E7	0.335	0.466	0.691	84.08％
					2E5+2C2	0.335	0.508	0.738	85.77％

The experimental results are shown in tables 1, 2E5 and 11C12 (the antibody sequence is disclosed in SEQ ID NO.1 of CN 106946989A), 15E7 (the antibody sequence is disclosed in antibody VHH-CEA 1 of CN106749667A, SEQ ID NO.4) and 2C2 (the antibody sequence is disclosed in SEQ ID NO.4 of CN 106946989A) which are respectively directed against different epitopes of CEACAM-5 antigen, which indicates that in the diagnosis and treatment application of single domain antibody, 2E5 single domain antibody can be jointly applied as a composition for complementing the epitopes of other single domain antibodies, thereby increasing the diagnosis or treatment efficiency.

Example 6 analysis of Single Domain antibody 2E5 binding site Using Biacore

The principal principle of the Biacore system is that SPR (refractive index) shifts by changes in the concentration of surface molecules, which appear on the monitor as changes in RU. Due to the higher sensitivity of the system, we designed relevant experiments to verify the ELISA-superimposed experimental results. As shown in fig. 4, first repeating 2 needles of the first single domain antibody a, observing changes in RU values to confirm saturation of the corresponding antigen binding sites and recording; then into the second single domain antibody B, RU values were observed and recorded: if the RU value is not more than 20% different from the RU value of single domain antibody B, the two can be considered to recognize different epitopes; if the difference is more than 20% but less than 60%, the two are considered to have steric hindrance; if the difference value exceeds 60%, the two are judged to recognize the same antigen. The specific operation is that firstly, the increased value R of RU is recorded by the antibody B which is injected only_B1And regenerating the chip; antibody A was then repeated twice and RU increase value R was recorded_AAnd after confirming saturation, directly injecting an antibody B, and observing the increase R of RU value_B2(ii) a Then using the formula (R)_B2-R_A)/R_B1The steric hindrance is calculated to determine whether both recognize the same epitope. The results of this example are shown in Table 2. 2E5 single domain antibody and 11C12 (the antibody sequence is disclosed in SEQ ID NO.1 of CN 106946989A), 15E7 (the antibody sequence is disclosed in CN 10674966)The steric hindrance of the antibody VHH-CEA 1 of 7A, SEQ ID NO.4), 2C2 (the sequence of this antibody is published in SEQ ID NO.4 of CN 106946989A) three-strain single-domain antibody was 5.79%, 17.98% and 13.64%, respectively. The results of judging that 2E5 and other three single-domain antibodies recognize different antigenic sites sequentially are consistent with the results presumed by the ELISA overlapping experiment. The application prospect of the anti-CEACAM-5 single domain antibody 2E5 in the field of diagnosis and treatment of CEA is further verified.

Table 2: RU value change table for Biacore detection single-domain antibody superposition experiment

Example 72 application of E5-HAP in detecting CEACAM-5 content in serum of clinical samples

The amino acid sequence of the binding site sequence of human alkaline phosphatase as the chemical light emitting region is shown as SEQ ID NO. 8. The fusion of the flexible polypeptide and the 2E5 single-domain antibody is carried out to obtain the single-domain antibody 2E5-HAP with a chemical light-emitting region sequence, and the amino acid sequence of the single-domain antibody is shown as SEQ ID NO. 7. Two restriction sites HindIII and EcoRI were added to the two ends of the nucleotide coding sequence and ligated to the vector pcDNA3.1 (+). After endotoxin-free large-scale plasmid extraction, 293 cells in logarithmic growth were used for transfection. After the transfected cells are cultured for 36h, the cell culture solution is poured into a 50ml centrifuge tube, 12000g is centrifuged for 5min, the supernatant is collected, filtered by a 0.22um filter membrane, and the culture supernatant is purified by anion exchange chromatography. The affinity test of 2E5-HAP was carried out in the same manner as in example 3, and the affinity value of 2E5-HAP was 9.173E-11. The results of the screening and matching are shown in Table 3. The FC fusion single domain antibody 2C2 (the antibody sequence is disclosed in SEQ ID NO.4 of CN 106946989A) is selected as a capture primary antibody, the variable region sequence of the FC fusion single domain antibody contains an amino acid sequence shown in SEQ ID NO.6, 2E5-HAP is an enzyme-labeled secondary antibody, and a double-antibody sandwich immunoassay method is carried out to detect the CEACAM-5 antigen in a serum sample, so that an excellent detection effect is obtained, and the specific process is as follows:

diluting FC fusion single domain antibody 2C2 with sterile CBS to a final concentration of 10 μ g/ml; adding 100 mul of the enzyme-linked immunosorbent assay (ELISA) plate into each hole, and standing for 18h at 4 ℃; discarding the supernatant, adding 300 μ L of washing solution into each well, shaking horizontally for 3min, and absorbing and discarding the supernatant; the plate was washed four times. Mu.l of 1% BSA was added to each well and allowed to stand at 37 ℃ for 1 h. Washing the plate for four times; adding 50 mu L of positive control, negative control or sample to be detected into each hole; adding 50 mu L of freshly diluted enzyme-labeled secondary antibody (namely single-domain antibody 2E 5-HAP) into each well, diluting to the working concentration of 2 mu g/ml, and placing on a shaking table and shaking for 3-5 s; incubate at 37 ℃ for 1 h. Washing the plate for four times; adding 100 mu L of AP Chemiluminescence color development liquid (BM Chemiluminescence ELISA Substrate) into each hole, and shaking on a shaking table for 3-5 s; incubating for 10min at room temperature in dark; and selecting a microplate reader program Luminescence, measuring the Lum value of each hole and calculating the CEACAM-5 antigen value of the quality control serum. Results 2E5 and 2C2 single-domain antibody pair show the best matching result, positive serum samples are all detected to be positive, no missing detection condition exists, and the average value of the content of CEACAM-5 in the positive serum is 45.8 ng/ml. And the serum of the CEA sample shows a good linear curve, and R2 is 0.9934.

TABLE 3 result of CEACAM-5 antigen positive serum detection by single domain antibody 2E5-HAP and three single domain antibodies 11C12, 15E7 and 2C2

Sequence listing

<110> Changchun force Tai Biotechnology Limited

<120> anti-CEACAM-5 single domain antibody and application thereof

<160> 8

<170> PatentIn version 3.3

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Gly Arg Ser Phe Gly Thr Tyr

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

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Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

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Ile Ser Trp Ser Gly Gly Ser Ile Trp Tyr Ala Asp Ser Val Lys Gly

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Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

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Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala

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Tyr Asp Ser Gln Arg Ser Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser His

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Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

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Ala Gly Ile Ser Trp Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

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

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

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Asn Ala Ala Leu Ser Glu Arg Thr Pro Ile Ala Thr Met Pro Ser Gln

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Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser His

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Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

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Ala Gly Ile Ser Trp Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val

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

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

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Asn Ala Ala Leu Ser Glu Arg Thr Pro Ile Ala Thr Met Pro Ser Gln

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Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Gly

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Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Ile

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Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu

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Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys

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

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Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro Glu

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Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr

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Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala Thr Ala

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

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Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile

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Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val Val

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Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His

290 295 300

Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser Ala

305 310 315 320

Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met

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Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Arg Met

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Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly Gly Thr

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Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln

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Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu

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Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp Met

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Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu

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Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe

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

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

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Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu Val His

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Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe Ala Ala

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Thr Asp

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Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala

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Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala

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

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Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly

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Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser

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Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr Ala

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Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu

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Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu

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Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly

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Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr

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Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala

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Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser

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Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe

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Arg Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln Gly

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Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys

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Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala

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Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly

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Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu

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Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg

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Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His His

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Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp

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

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Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro

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Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp

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Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val

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

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Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His

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Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His Leu

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Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala Phe

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Gly Thr Thr Asp

Claims

1. A single domain antibody against CEACAM-5, characterized in that the variable region of said single domain antibody has 3 complementarity determining regions CDR1, CDR2, CDR3, wherein the CDR1 sequence consists of the amino acid sequence shown in SEQ ID No.1, the CDR2 sequence consists of the amino acid sequence shown in SEQ ID No.2, and the CDR3 sequence consists of the amino acid sequence shown in SEQ ID No. 3.

2. The single domain antibody of claim 1, wherein the variable region sequence of said single domain antibody consists of the amino acid sequence shown in SEQ ID No. 4.

3. A fusion protein of the single domain antibody of claim 2 and human alkaline phosphatase, wherein said fusion protein is formed by the tandem connection of said single domain antibody and human placental alkaline phosphatase.

4. A polynucleotide encoding the single domain antibody of claim 2, wherein the sequence of said polynucleotide is set forth in SEQ ID No. 5.

5. An expression vector comprising the polynucleotide of claim 4, wherein said vector is pMES 4.

6. A host cell comprising the expression vector of claim 5, wherein said cell is E.coli BL21(DE 3).

7. Use of a single domain antibody according to claim 1 or 2 in the preparation of a CEACAM-5 antigen detection kit.

8. A CEACAM-5 antigen immunodetection method based on a non-diagnosis purpose is characterized in that the method is a double-antibody sandwich enzyme-linked immunoassay, the amino acid sequence of a variable region of a primary antibody is shown as SEQ ID NO.6, a secondary antibody is an enzyme-linked secondary antibody, and the amino acid sequence of the variable region is shown as SEQ ID NO. 4.

9. The method of claim 8, wherein the enzyme-linked secondary antibody is a fusion protein of a single domain antibody against CEACAM-5 and human placental alkaline phosphatase.

10. An immunoassay kit for detecting CEACAM-5 antigen by using a double antibody sandwich method, the kit comprises a primary antibody for capturing the antigen and a secondary antibody for binding with the antigen to initiate an enzyme-linked reaction, and is characterized in that the amino acid sequence of a variable region of the primary antibody is shown in SEQ ID NO.6, the secondary antibody is a fusion protein of a single-domain antibody for resisting the CEACAM-5 antigen and human alkaline phosphatase, and the sequence of the fusion protein is shown in SEQ ID NO. 7.