CN111004328A - Nano antibody combination for detecting carcinoembryonic antigen by double antibody sandwich method - Google Patents

Abstract

The invention discloses a group of nano-antibody combinations for detecting carcinoembryonic antigen by a double-antibody sandwich method, wherein the combinations comprise an anti-carcinoembryonic antigen bivalent nano-antibody serving as a capture antibody and an anti-carcinoembryonic antigen monovalent nano-antibody serving as a detection antibody. The nanometer antibody combination of the carcinoembryonic antigen bivalent nanometer antibody and the carcinoembryonic antigen monovalent nanometer antibody provided by the invention has good matching degree, shows excellent P/N value, lowest detection limit and accuracy in the detection of CEA antigen, and can meet the detection of CEA in clinical samples.

Description

Nano antibody combination for detecting carcinoembryonic antigen by double antibody sandwich method

Technical Field

The invention discloses a nano antibody combination, and belongs to the field of immunology.

Background

Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA 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). CEA was originally classified as a protein expressed only in fetal tissues and has now been identified in several normal adult tissues. Overexpression of CEA is observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Thus, CEA has been identified as a tumor associated antigen. CEA 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, serum CEA levels have been used as clinical markers to diagnose and screen for cancer. Furthermore, CEA has also been used as a tumor marker, and immunological assays to measure elevated CEA in the blood of cancer patients have been used clinically for the prognosis and control of cancer.

More importantly, CEA has become a potentially useful tumor-associated antigen for targeted therapy. There have been reported 2 major approaches to cancer treatment using CEA-targeted immunotherapy. One method uses an anti-CEA antibody to elicit the lytic activity of immune cells, particularly by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEA-expressing tumor cells. Another approach is to specifically target CEA-expressing tumor cells by conjugating the anti-CEA antibody or antibody fragment to an effector molecule such as a drug, toxin, radionucleotide, immunomodulator or cytokine, to exert the therapeutic effect of the effector molecule.

Various monoclonal antibodies have been generated against CEA. Chester et al have isolated single chain anti-CEA 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-CEA 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 are thus referred to as heavy chain-only antibodies (HCAbs). The antigen binding site of such antibodies consists of one domain, called VHH region, and thus such antibodies are 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.

Nanobodies 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.

The nanobody has the smallest functional antigen-binding fragment derived from HCabs in adult camelids, has high stability and high affinity for antigen binding, and can interact with protein cleft and active sites of enzyme, making its action similar to that of inhibitors. Therefore, the nano-antibody can provide a new idea for designing small molecule enzyme inhibitors from peptide-mimetic drugs. Due to the heavy chain only, nanobodies are easier to manufacture than monoclonal antibodies. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, allow for large yields to be produced at low cost. Therefore, the nano 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 CEA is more over-expressed in some solid tumors such as colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatoma, breast cancer and thyroid cancer, the development of anti-CEA nanobodies is a new need in the field of antibody technology to fully utilize the super-strong antigen recognition capability of nanobodies, and particularly to recognize some epitopes hidden in crevices or cavities. However, the existence of some structural defects such as low affinity, easy aggregation, short serum half-life and the like due to the low molecular weight of the nanobody prevents the further application of the nanobody.

At present, research and development reports and related patent applications of anti-CEA nano antibodies exist, and Chinese invention patent CN106749667A discloses three strains of anti-CEA nano antibodies which all have very strong antigen affinity, wherein the antigen affinity of VHH-CEA1 reaches 2.40E-9. Another Chinese patent application CN107880130A discloses a nano antibody 2D5 for resisting CEA, and the antigen affinity thereof reaches 2.67E-11. The excellent antigen affinity ensures the application prospect of the nano antibody in the differential diagnosis of the target antigen, but in the specific application, even the nano antibody with high antigen affinity still has great technical problems in the actual operation of antigen detection. For example, the overlapping degree of the antigen binding sites between the first antibody and the second antibody in the immuno-sandwich assay and the series of problems caused by the small molecular weight of the nano-antibody are still the technical problems of obtaining a high-efficiency detection kit. The invention aims to provide an anti-CEA nano antibody combination which can not only give full play to the superior performance of a nano antibody, but also overcome the inherent defects of the nano antibody so as to give full play to the high-efficiency performance of the nano antibody in CEA antigen detection.

The invention content is as follows:

based on the above objects, the present invention firstly provides a set of nanobody combinations for detecting carcinoembryonic antigen (CEA) by a double antibody sandwich method, wherein the combination comprises a bivalent nanobody against carcinoembryonic antigen as a capture antibody and a monovalent nanobody against carcinoembryonic antigen as a detection antibody, the variable region sequence of the bivalent nanobody is shown as SEQ ID No.1, and the variable region sequence of the monovalent nanobody is shown as SEQ ID No. 2.

In a preferred embodiment, the bivalent nanobody is linked to the variable region of the bivalent nanobody by both ends of one antibody hinge region, respectively.

In a more preferred embodiment, the amino acid sequence of the bivalent nanobody is shown in SEQ ID NO. 3.

In another preferred embodiment, the monovalent nanobody is linked to a chromogenic group.

More preferably, the chemochromic group is human alkaline phosphatase.

Particularly preferably, the monovalent nanobody is linked to the human alkaline phosphatase in a fusion expression manner.

The invention further provides application of the nano antibody combination in preparation of a carcinoembryonic antigen detection kit.

In a preferred embodiment, the detection kit is a magnetic bead chemiluminescence immunoassay kit.

More preferably, the bivalent nanobody is a biotinylated bivalent nanobody, and the kit further comprises streptavidin-based magnetic beads.

The nano antibody combination for detecting the carcinoembryonic antigen by the double-antibody sandwich method provided by the invention shows high-efficiency performance in CEA antigen detection, wherein the anti-carcinoembryonic antigen bivalent nano antibody serving as the capture antibody has double antigen binding sites of the monovalent nano antibody, and has higher recognition and binding capacity on the CEA antigen compared with the monovalent nano antibody. After the optimization and debugging of the detection reaction system, the reactivity, the lowest detection limit and the accuracy are greatly improved. The nanometer antibody combination of the carcinoembryonic antigen bivalent nanometer antibody and the carcinoembryonic antigen monovalent nanometer antibody provided by the invention has good matching degree, shows excellent P/N value, lowest detection limit and accuracy in the detection of CEA antigen, and can meet the detection of CEA in clinical samples.

Drawings

FIG. 1 shows the first round of PCR amplification and electrophoretic identification chart;

FIG. 2 is a second round of PCR amplification and electrophoresis identification chart;

FIG. 3 is a SDS-PAGE graph of bivalent nanobody purification;

FIG. 4 is a graph of a bivalent nanobody affinity test;

FIG. 5 is a graph of affinity test of biotinylated bivalent nanobody.

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 protection defined by the claims.

EXAMPLE 1 preparation of anti-CEA monovalent Nanobodies

Refer to chinese patent application CN 106749667A. The variable region sequence of the monovalent nanometer antibody is shown as SEQ ID NO. 1.

Example 2 preparation of anti-CEA bivalent Nanobody

2.1VHH1-LHC-VHH1-pMES4 vector construction:

two downstream primers were designed due to the longer antibody hinge region (LHC) and two rounds of PCR were performed as follows:

2.1.1 first round PCR was performed with monovalent nanobody DNA as template, and the primer sequences were as follows:

11F1：AACTGCAGGAGAGCGGTGGCGGTC

11R1:CAAGTGACCGTTAGCAGCGAACCGAAAACCCCGAAACC

GCAGCCGCAGCCTCAACCGCAACCGCAGCCG

the PCR reaction conditions and procedures were: 5 minutes at 95 ℃; 30 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 30 seconds; 7 minutes at 72 ℃. The agarose gel recovery kit gel was used to recover a band of about 300bp (FIG. 1: M is Trans 2K DNAmarker; 1 is a negative control; 2-10 is a first round PCR product).

2.1.2 second round PCR was performed using the recovered product of the first round PCR as a template, and the primer sequences were as follows:

11F1:AACTGCAGGAGAGCGGTGGCGGTC

11R2:CAACCGCAACCGCAGCCGAATCCGACCACCGAAAGCA

AAGCAAATGTCCGCACCACCATCACCATCACTAGTC

the PCR reaction conditions and procedures were: 5 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃ and 15 cycles; 7 minutes at 72 ℃. PCR products were purified using the PCR product recovery kit (FIG. 2: M is Trans 5K DNAmarker; 1-3 are second round PCR products; 4 is a negative control).

2.1.3 vector construction: pMES4 (from Biovector) and the second PCR product were digested simultaneously with Pst I and SpeI, respectively, and 1.5. mu.g of the digested vector and 450ng of the digested second PCR product were ligated overnight at 16 ℃ with 1.5. mu. L T4 DNA ligase supplemented with buffer and water to a total volume of 10. mu.L.

2.2 induction expression of bivalent nanometer antibody;

2.2.1VHH1-LHC-VHH1 ligation product transformation of E.coli Shuffle: the ligation product 10ul was transformed into 100 ul Shuffle competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a water bath at 42 ℃ for 90 seconds, and cooled in an ice bath for 3 minutes. 600. mu.l of LB medium was added to the centrifuge tube, and the tube was cultured with shaking at 37 ℃ for 60 minutes. 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.

2.2.2 Induction expression and extraction of bivalent Nanobody: and (3) selecting the 10 monoclonal colonies, performing shaking culture at 37 ℃ overnight in an LB-A culture medium, sequencing, and referring to SEQ. NO.3 or 4 for a correct sequence. Strains with correct sequencing are selected for inducible expression. Taking the bacterial liquid according to the proportion of 1: adding 100ml of fresh LB-A culture medium in a proportion of 100, and performing shaking culture at 37 ℃ for 3 hours 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, the cells were collected by centrifugation for 10 minutes, and 1.5mL of precooled TES buffer was added to resuspend the pellet. After 2 minutes in ice bath, gently shake for 30 seconds and repeat this cycle 6 times. 3.0ml TES/4 (TES diluted 4-fold with water) was added, gently shaken for 30 seconds, and then allowed to stand on an ice bath for 2 minutes, and the shaking and standing steps were repeated a total of 6 times. Centrifuging at 9000rpm and 4 deg.C for 10 min, and collectingAbout 4.5mL of the supernatant (periplasmic extract) was subjected to protein electrophoresis.

2.2.3 purification and characterization of bivalent nanobodies: after resuspending IMAC Sepharose (GE Co.), 2ml was added to the gravity column, and the column was allowed to stand for 30 minutes to allow Sepharose to naturally settle at the bottom of the gravity column, and the preservation buffer was discharged. Adding 2 column volumes of nickel sulfate solution (0.1M) and flowing out the nickel sulfate solution at a flow rate of about 8 seconds per 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 seconds/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 being 3 times of that of the column, maintaining the flow rate at 6 seconds per drop, and collecting the eluent containing the target protein; finally sepharose was washed by sequentially adding 10 column volumes of equilibration buffer, 10 column volumes of pure water and 10 column volumes of 20% ethanol, and finally 4ml of 20% ethanol was retained to preserve the column. The collected samples were subjected to SDS-PAGE detection (FIG. 3: M is rainbow 180 broad-spectrum protein; 1 is VHH1-LHC-VHH1, which is a purified nano-antibody induced by E.coli). The amino acid sequence of the bivalent nano antibody is shown as SEQ ID NO.3, and the nucleotide coding sequence thereof is shown as SEQ ID NO. 4.

Example 3 affinity testing of anti-CEA Nanobodies

3.1 chip antigen coupling: preparing the CEA antigen into working solution of 20 mu g/mL by using sodium acetate buffer solutions (pH 5.5, pH 5.0, pH 4.5 and pH 4.0) with different pH values, preparing 50mM NaOH regeneration solution, analyzing the electrostatic binding between the antigen and the surface of a chip (GE company) under different pH conditions by using a template method in a Biacore T100 protein interaction analysis system instrument, selecting a proper pH system with most neutral pH according to the standard that the signal increase amount reaches 5 times RL, and adjusting the antigen concentration as the condition during coupling according to the requirement. 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 minutes.

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 injection conditions were 120 seconds and 30. mu.L/min. Regeneration conditions were 30 seconds, 30. mu.L/min. The buffer was run continuously empty first until all baselines were stable. The nanobody solution with larger concentration span is prepared to be configured with the running buffer, and 200. mu.g/mL, 150. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL and 2. mu.g/mL are suggested to be set. 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 nano antibody and the antigen is tested by using a template method carried by the instrument (wherein the sample introduction condition is set to be 60 seconds and 30 mu L/min; the dissociation time is 600 seconds, and the regeneration condition is set to be 30 seconds and 30 mu L/min). The signal condition of the 2-1 channel is observed at any time. The affinity testing process took approximately 200 minutes. In a specific experiment, nanobodies on the chip were captured to the appropriate signal values and then injected onto the chip with system running buffer HBS-EP (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20) at a flow rate of 30 μ L/min to obtain a dynamic process of nanobody-antigen interaction. The ability of monovalent/bivalent nanobodies to bind to and dissociate from antigens was tested using this method, respectively.

3.4 analysis of results: the binding dissociation curves for several concentration gradients were selected using a 1: all curves are fitted in the 1binding mode, and important parameters such as affinity values, binding constants and dissociation constants are finally obtained (see fig. 4). The affinity of the screened univalent/bivalent nano antibody reaches 10^-9The above.

Table 1: monovalent/divalent nanobody affinity data

Sample numbering	Affinity of
		VHH-CEA1	2.40E-09
VHH1-LHC-VHH1	3.08E-10

Example 4 preparation of biotinylated anti-CEA Nanobodies

Coupling of monovalent/bivalent nanobodies to biotin was performed using a biotinylated coupling kit purchased from Thermo, with reference to the instructions, as follows:

biotin was first diluted to 10mM with ultrapure water while ensuring that the antibody was stored in PBS or buffer PH 7.2-8.0; according to the calculation formula provided in the specification, as follows,

mixing the nano antibody and biotin according to a calculated proportion, and coupling at room temperature for more than 30 min; and a purification step, namely purifying by using a nickel column to obtain 30ml of eluent, and further purifying by using a molecular sieve. The affinity test was performed on biotinylated monovalent/bivalent nanobodies according to the method of example 3 (see results in fig. 5), and the values are shown in table 2:

TABLE 2 biotinylated monovalent/divalent Nanobody affinity data

Name (R)	Affinity number
		Bio-VHH-CEA1	3.12E-09
Bio-VHH1-LHC-VHH1	1.05E-08

Example 5.2 preparation of D5-HAP

Referring to the disclosure of the specification of Chinese patent application CN107880130A, the variable region sequence of the nano antibody 2D5 is shown as SEQ ID NO. 2. The patent application discloses the fusion of nanobody with human alkaline phosphatase into nanobody 2D5-HAP with chemoluminescent region sequence by flexible polypeptide. 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 nanobody with a chemiluminescent region was subjected to an affinity test using the same method as in example 3, and the 2D5-HAP affinity value was 2.67E-11.

Example 6 application of anti-CEA Nanobody in detection kit

Selecting a biotinylated nano antibody (bivalent/monovalent) as a capture antibody and 2D5-HAP as a detection antibody, and carrying out a double-antibody sandwich immunoassay method to detect the CEA antigen in a serum sample, wherein the method uses a magnetic bead chemiluminescence method, and comprises the following specific processes:

uniformly mixing 80 ul/hole of biotinylated nano antibody (1ug/ml), 30 ul/hole of CEA quality control product (10ng/ml of Roche chemiluminescence CEA diagnostic kit quality control product) or negative serum and 80 ul/hole of 2D5-HAP (3ug/ml), placing in a 96-hole microporous plate, and incubating at 37 ℃ for 15 min; washing, adding washing solution at 300 ul/hole, mixing, standing on magnetic frame for 3min, removing supernatant, and repeating the above washing steps for 4 times; adding streptavidin magnetic beads (purchased from JSR) (0.3mg/ml)80 ul/well, mixing, and incubating at 37 ℃ for 15 min; repeating the above washing for 5 times; adding 100 mu L/well of AP chemiluminescent chromogenic solution (BM Chemiluminescence assay) and shaking on a shaking table for 3-5 s, selecting a microplate reader program luminence, and measuring the reading value of each well.

6.1 detection of P/N: the ratio of the detection value of the positive quality control substance (10ng/ml) to the detection value of the negative serum.

6.2 detection of the lowest detection limit: and (3) detecting by using a zero-concentration calibrator or a sample diluent as a sample, repeatedly measuring for 20 times, calculating the numerical value of the measurement result of 20 times, and calculating the average value (M) and the Standard Deviation (SD) of the numerical value to obtain M +2SD, namely the lowest detection limit.

6.3 detection of accuracy (recovery): adding carcinoembryonic antigen (CEA) liquid (A) with the concentration of about 50ng/ml (allowable deviation +/-10%) into serum B with the concentration range of 2 ng/ml-5 ng/ml, wherein the volume ratio of the CEA to the serum B is 1:9, and calculating the result according to the formula (1);

in the formula: r- - -recovery;

v- - -adding the volume of the solution A;

v0 — volume of serum sample B;

c- -the detection concentration of the serum sample after the solution A is added;

c0-concentration of serum sample B to be detected;

concentration of CS- - - -A solution

6.4 referring to Chinese invention patent CN106749667A, antigen-antibody epitope overlapping tests were performed on CEA1, CEA2 and CEA3, and the data are shown in Table 3:

TABLE 3 antigen antibody epitope overlay assay

Result analysis shows that the superposition rate AI of the two antibodies is more than 50 percent, which indicates that the antigenic sites of the 2 antibodies are different, the AI is less than 50 percent, which indicates that the antigenic epitopes of the two antibodies are the same, and the larger the AI value is, the lower the possibility of site overlapping is. The above data illustrate that VHH-CEA1+ VHH-CEA3 is the best combination.

6.5 referring to the Chinese invention patent CN107880130A, VHH-CEA1 as capture antibody, VHH-CEA3-HAP and 2D5-HAP as detection antibody for pairing detection, the results show that VHH-CEA1+2D5-HAP has excellent detection effect, and VHH-CEA1 and 2D5 are the best combination.

6.6 this patent is directed to the optimal combination of VHH-CEA1 and 2D5 for bivalent nanobody construction and biotinylation, paired detection, data shown in Table 4.

TABLE 4 univalent/bivalent Nanobody pairing assay results

The results show that the P/N value, the lowest detection limit and the accuracy of the reaction of the bivalent nano antibody Bio-VHH1-LHC-VHH1 and 2D5-HAP are superior to those of the univalent nano antibody and other types of nano antibody combinations.

Sequence listing

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<120> a nano-antibody combination for detecting carcino-embryonic antigen by double-antibody sandwich method

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Claims (9)

1. A group of nanometer antibody combinations for detecting carcinoembryonic antigen by a double-antibody sandwich method comprises an anti-carcinoembryonic antigen bivalent nanometer antibody used as a capture antibody and an anti-carcinoembryonic antigen monovalent nanometer antibody used as a detection antibody, wherein the variable region sequence of the bivalent nanometer antibody is shown as SEQ ID NO.1, and the variable region sequence of the monovalent nanometer antibody is shown as SEQ ID NO. 2.

2. The nanobody combination according to claim 1, wherein the bivalent nanobody is linked to the variable region of the bivalent nanobody by both ends of one antibody hinge region, respectively.

3. The nanobody combination according to claim 2, wherein the amino acid sequence of the bivalent nanobody is shown in SEQ ID No. 3.

4. The nanobody combination of claim 1, wherein the monovalent nanobody is linked to a chemochromic group.

5. The nanobody combination of claim 4, wherein the chemochromic group is human alkaline phosphatase.

6. The nanobody combination according to claim 5, wherein the monovalent nanobody is linked to human alkaline phosphatase in a fusion expression linkage.

7. Use of the nanobody combination of any one of claims 1 to 6 for the preparation of a carcinoembryonic antigen detection kit.

8. The use of claim 7, wherein the detection kit is a magnetic bead chemiluminescence immunoassay kit.

9. The use according to claim 8, wherein the bivalent nanobody is a biotinylated bivalent nanobody and the kit further comprises streptavidin-coated magnetic beads.

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