CN108593912A - A kind of detection method of solubility CD38 concentration - Google Patents

Abstract

The invention relates to a method for detecting the concentration of soluble CD38. A pair of single-domain antibodies capable of simultaneously binding to CD38 was screened, that is, single-domain antibody 1053 and 551, wherein single-domain antibody 551 was used as a capture antibody, and the fusion protein of single-domain antibody 1053 and firefly luciferase mutant Fluc2 was used as a detection antibody. The single-domain antibody 551 was coated on the bottom of the ELISA plate to capture CD38 in the solution, and the luminescence signal of Fluc2 in the antibody catalyzed by luciferin was detected to evaluate the concentration of CD38, and a specific and ultra-sensitive soluble CD38 detection method was developed .

Description

A method for detecting the concentration of soluble CD38

technical field

The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to a method for detecting the concentration of soluble CD38.

Background technique

CD38 is a single transmembrane glycoprotein that is often used as a marker of cell differentiation. In 1996, soluble CD38 (soluble CD38, sCD38) was discovered for the first time, which was considered to be the product released by cleavage of the extramembrane domain of the full-length CD38 molecule. They exist in a variety of physiological and pathological body fluids and tumor cell culture supernatants, and retain the antigenic properties and catalytic activity of CD38 ¹ . Subsequent studies have found that soluble CD38 can act as a signaling molecule to induce cell proliferation and migration2, prolong the lifespan ^of memory antibodies3 and regulate maternal ^- fetal tolerance4 and other functions ^.

CD38 is abnormally highly expressed ^on the surface of various blood cancer cells, such as multiple myeloma cells, while it is in a low expression state on normal lymphocytes, bone marrow cells and some non-hematopoietic cells. Therefore, CD38 is considered to be a target for the treatment of multiple myeloma, and multiple therapeutic monoclonal antibodies against it are under ^development6 . The expression of CD38 on the cell surface is a diagnostic indicator for multiple myeloma, but the acquisition of myeloma cells requires bone marrow aspiration, which is invasive, and local extraction of bone marrow cannot reflect the overall myeloma burden of the patient, so blood diagnosis Has superiority.

Since the patient's plasma is a complex system, the content of soluble CD38 is very low, and a highly sensitive method is required for accurate detection. At present, a CD38 detection ELISA kit with the highest detection sensitivity on the market uses traditional antibodies for solid-phase sandwich enzyme-linked immunosorbent assay, and its detection sensitivity is nominally 93.75pg/mL. Disadvantages: 1) The detection sensitivity is still limited; 2) The detection specificity is not strong, and it is not suitable for detecting complex samples; 3) The cost is high, and a kit of 5,000 yuan can only do 96 samples/test.

references

1 Funaro, A. et al. Identification and characterization of an active soluble form of human CD38 in normal and pathological fluids. International Immunology 8, 1643-1650 (1996).

2 Deaglio, S. et al. CD38/CD31 interactions activate genetic pathways leading to proliferation and migration in chronic lymphocytic leukemia cells. Molecular medicine 16, 87-91, doi: 10.2119/molmed. 2009.00146(2010).

3 Liu, X.Q., Hart, D.N., MacPherson, G.G., Good, M.F.&Wykes, M.N. Soluble CD38 significantly prolongs the lifespan of memory B-cell responses. Immunology 125, 14-20, doi: 10.1111/j. 2008).

4 Kim, B.J.et al.Seminal CD38 is a pivotal regulator for fetomaternal tolerance.Proceedings of the National Academy of Sciences of the United States of America 112,1559-1564,doi:10.1073/pnas.1413493112(2015).

5 Lin, P., Owens, R., Tricot, G. & Wilson, C.S. Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma. American journal of clinical pathology 121, 482-488, doi: 10.1309/74R4-TB90-BUWH.X204JWH-27

6 van de Donk, N., Richardson, P.G. & Malavasi, F. CD38 antibodies inmultiple myeloma: back to the future. Blood 131, 13-29, doi: 10.1182/blood-2017-06-740944 (2018).

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for detecting soluble CD38 with ultra-high sensitivity and specificity, and capable of high-throughput detection.

To achieve the above purpose, the first aspect of the present invention provides a detection method for soluble CD38, that is, a sandwich method, including a capture antibody and a detection antibody.

The second aspect of the present invention provides a capture antibody and a detection antibody, that is, CD38 single domain antibody and CD38 single domain antibody luciferase fusion protein, including CD38 single domain antibody part and firefly luciferase part.

The third aspect of the present invention provides two DNA molecules, which encode the capture antibody and detection antibody of the present invention, or the CD38 single domain antibody luciferase fusion protein of the present invention.

In the fourth aspect of the present invention, two expression vectors are provided, which comprise the gene sequence of the CD38 single domain antibody and the gene sequence of the firefly luciferase mutant.

In the fifth aspect of the present invention, two kinds of host cells are provided, which contain the aforementioned expression vectors.

The CDS sequence of the gene encoding the CD38 single domain antibody 1053 of the present invention has a full length cDNA sequence of 546bp (sequence 1), and the specific sequence is as follows:

ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCGATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACAGGCTCAGGACGCACCTTCAGGAACTATCCCATGGCCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTGTAGCAGGTATTACCTGGGTCGGTGCTAGCACACTCTATGCAGACTTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATAGTTGTGCAGCAGGTCGCGGTATAGTGGCTGGTAGGATCCCAGCTGAGTATGCCGACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAACCAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAG

The encoding produces a protein sequence (sequence 2) with a length of 181 amino acids (the terminal terminator is not included, that is, the number *** in the sequence is not included), and the specific sequence is as follows:

The CDS sequence of the gene encoding the CD38 single domain antibody 551 of the present invention has a full length cDNA sequence of 558bp (sequence 3), and the specific sequence is as follows:

ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCGATGTGCAGCTGCAGGAGTCAGGAGGAGGATTGGTGCAGGCTGGACACTCTCTGAGACTCTCCTGTGTAGGCTCCGGTAGCAGATTCGATAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCCGCTATTAGCTGGAGTAGTGGCACTACGCGCTATTTAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGTACGGTATATCTTCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTCGATATCAGCCGAGGTACTACGACTCAGGGGATATGGATGGATATGAGTATGACAACTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAACCAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAG

The encoding produces a protein sequence (sequence 4) with a length of 185 amino acids (the terminal terminator is not included, that is, the number *** in the sequence is not included), and the specific sequence is as follows:

The CDS sequence of the gene encoding the detection antibody of the present invention has a cDNA sequence of 2085 bp in full length (sequence 5), and the specific sequence is as follows:

TCCGGTACCATGGATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACAGGCTCAGGACGCACCTTCAGGAACTATCCCATGGCCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGTGAGTTTGTAGCAGGTATTACCTGGGTCGGTGCTAGCACACTCTATGCAGACTTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATAGTTGTGCAGCAGGTCGCGGTATAGTGGCTGGTAGGATCCCAGCTGAGTATGCCGACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAACCAGCGGAGCTCCCGGGGGCGGCCGCCTGCAGAATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCG ATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCT TCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGG ACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGTGA

The encoding produces a protein sequence (SEQ ID NO: 6) with a length of 694 amino acids (the terminal terminator is not included, that is, the number *** in the sequence is not included), and the specific sequence is as follows:

Implementing the present invention has the following beneficial effects: the present invention provides a method for detecting soluble CD38 with ultrahigh sensitivity and specificity and high throughput, and its sensitivity reaches 10pg/mL, which is higher than commercial ELISA kits; more importantly, relatively In the kit, the DepID method can effectively detect the content of CD38 in complex samples (such as plasma samples).

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the drawings used in the description of the embodiments are briefly introduced. In the attached picture:

Figure 1 is the SDS-PAGE analysis diagram of CD38 single domain antibody 1053, 551 and recombinant protein 1053-Fluc2;

Figure 2 is a competition diagram of CD38 binding between single domain antibody 1053, 551, 375, NbGFP and labeled single domain antibody 551-AF48;

Figure 3 is a competition diagram of CD38 binding between single domain antibody 1053, 551, 375, NbGFP and labeled single domain antibody 1053-AF488;

Fig. 4 is the detection schematic diagram of DepID method;

Fig. 5 is the standard curve of CD38 detected by DepID method;

Figure 6 is a diagram of the detection specificity of the DepID method;

Figure 7 is a comparison of soluble CD38 levels in plasma of healthy volunteers and multiple myeloma patients detected by DepID.

Detailed ways

Embodiments of the present invention will be specifically described below in conjunction with the accompanying drawings.

Since soluble CD38 is derived from CD38 on the cell surface, the concentration of soluble CD38 in plasma can reflect the proliferation of multiple myeloma cells and the activity of Sheddase in the tumor microenvironment, which can be used for diagnosis and monitoring of multiple myeloma disease development of markers.

The present invention describes a specific and ultra-sensitive soluble CD38 detection method developed based on two single-domain antibodies that bind to different epitopes of CD38—Dual e pitopes protein ID entification , DepID), and showed a correlation between soluble CD38 and the course of multiple myeloma.

In the present invention, a series of CD38 single-domain antibodies screened are screened out by flow cytometry to single-domain antibody pairs that can simultaneously bind to CD38, that is, single-domain antibodies 1053 and 551, and single-domain antibody 551 is used as a capture antibody. Fusion protein of single domain antibody 1053 and firefly luciferase mutant Fluc2 was used as detection antibody. The single-domain antibody 551 was coated on the bottom of the ELISA plate to capture CD38 in the solution, and the luminescence signal of Fluc2 in the antibody catalyzed luciferin was detected to evaluate the concentration of CD38 (as shown in Figure 4). Highly sensitive soluble CD38 detection method DepID.

Below in conjunction with specific embodiment, further illustrate the present invention.

Example 1: Construction of DepID detection antibody expression vector

(1) The gene sequence of CD38 single domain antibody 1053 was amplified by PCR, and the pRHSUL2 vector and 1053 gene sequence were digested with restriction enzymes KpnI and SacI (purchased from Thermo Scientific), and ligated with T4 DNA ligase (purchased from TAKARA Company) Two fragments, pRHSUL2-1053 were constructed.

(2) Digest the pRHSUL2-1053 vector with restriction endonucleases SacI and PstI, and anneal the two DNA single strands containing SacI and PstI cohesive ends and 2×G ₄ S sequence, and use T4 DNA ligase to separate the 2×G ₄ S was inserted into pRHSUL2-1053 to construct pRHSUL2-1053-G ₄ S.

(3) PCR amplifies the gene sequence of the firefly luciferase mutant Fluc2, uses restriction endonucleases PstI and HindIII to digest the gene sequence of pRHSUL2-1053-G ₄ S and Fluc2, and uses T4 DNA ligase to connect the two fragments, Construct pRHSUL2-1053-G ₄ S-Fluc2, which is the expression vector of DepID detection antibody.

Example 2: Expression and purification of CD38 single domain antibody and DepID detection antibody 1053-Fluc2

(1) Transform the CD38 single domain antibody expression vectors pHEN2-1053, pHEN2-551 and the recombinant plasmid pRHSUL2-1053-G ₄ S-Fluc2 identified by sequencing into the expression host strain Rosetta2 (DE3), and expand the culture to OD at 37°C When the ₆₀₀ reaches 0.6-0.8, add 1mM IPTG to induce expression at 18°C, collect the bacteria by ultrasonication, and collect the supernatant by high-speed centrifugation for further purification.

(2) CD38 single domain antibody was purified by Ni-NTA affinity chromatography and anion exchange chromatography to obtain purified protein.

(3) After the DepID detection antibody is purified by Ni-NTA, use sumo proteinase to remove the 6×HisSumo-tag, and then go through Ni-NTA reverse phase chromatography once, collect the effluent and perform Q column exchange chromatography to obtain the recombinant protein 1053-Fluc2. The SDS-PAGE results of single domain antibodies 1053, 551 and recombinant protein 1053-Fluc2 are shown in Figure 1.

Example 3: CD38 single domain antibody labeling

(1) Ultrafiltration of the CD38 single domain antibody, replacing the solution with a Tris-free buffer such as PBS;

(2) Alexa Fluor ^TM 488 succinyl ester (ThermoScientific), a fluorescent dye that can react with amino groups, was dissolved in DMSO;

(3) Add about 5 times the molar amount of the above-mentioned fluorescent dye to the CD38 single domain antibody, and rotate and mix overnight at 4°C in the dark;

(4) Ultrafiltration to remove the unlabeled fluorescent dye in the CD38 single domain antibody solution.

Example 4: CD38 single domain antibody pair selection

(1) Count the multiple myeloma cell line LP-1, adjust the cell density to 5×10 ⁵ /mL, and wash twice with pre-cooled PBS (containing 1 mg/mL BSA);

(2) Add 0.5 μg/mL labeled single domain antibody and gradient concentration of single domain antibody, and incubate at 4°C in the dark for 30 minutes;

(3) Wash twice with pre-cooled PBS (containing 1 mg/mL BSA), and resuspend in 100 μL PBS (containing 1 mg/mL BSA);

(4) Flow cytometry detection, the detection results are shown in Figure 2 and Figure 3, single domain antibodies 1053 and 551 can bind to different epitopes of CD38 without affecting each other.

Embodiment 5: DepID method steps

(1) Single domain antibody 551 was diluted to 10 μg/mL with PBS, 100 μL per well was coated on an ELISA plate, and left overnight at 4°C;

(2) Wash the plate three times with PBS;

(3) Dilute 1% BSA in 0.1% PBST (PBS, 0.1% Tween 20), 100 μL per well, block at room temperature for 1 h;

(4) Wash three times with 0.1% PBST;

(5) Dilute 20 μL of CD38 standard or sample with 0.5 μg/mL 1053-Fluc2 and 1 mg/mL BSA in 0.1% PBST to 100 μL, add to the ELISA plate, and incubate at room temperature for 1 h;

(6) Wash three times with 0.1% PBST;

(7) Add luciferin substrate for dynamic detection, take the initial 15s average value for calculation, the standard curve for detecting CD38 is shown in Figure 5, and its sensitivity can reach 10pg/mL.

Example 6: DepID specific detection

(1) Take out the CD38 single domain antibody beads and wash them twice with PBS;

(2) CD38 standard substance or plasma sample, each sample was divided into two equally, one of which was added with CD38 single domain antibody beads and incubated at 4°C for 3 hours, then centrifuged to remove the supernatant;

The samples treated with CD38 single-domain antibody beads and untreated samples were detected according to Example 5, and the specificity results are shown in Figure 6. After CD38 in the sample was removed by the single-domain antibody beads, the detection signal decreased to the background level. At the same time, the plasma samples of healthy volunteers and multiple myeloma patients were tested. As shown in Figure 7, the plasma soluble CD38 of the patients was significantly higher than the normal level. In addition, when comparing the DepID method with the commercial ELISA kit, the patient's plasma sample was detected at the same time. Table 1 is the comparison of the results of the DepID method and the commercial ELISA kit for the simultaneous detection of the soluble CD38 level in the patient's plasma sample.

Table 1

N.D.: Not detectable (not detectable).

The results showed that DepID can effectively detect the concentration of soluble CD38 in plasma, while commercial ELISA kits did not detect the signal, fully demonstrating the specificity of DepID method, especially suitable for the detection of complex samples.

Embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementations, and the above-mentioned specific implementations are only illustrative, rather than restrictive, and those of ordinary skill in the art will Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.

sequence listing

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<120>A method for detecting the concentration of soluble CD38

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ATGGCCGATG TGCAGCTGCA GGAGTCTGGA GGAGGCTTGG TGCAGGCTGG GGGCTCTCTG 120

AGACTTCTCCT GTACAGGCTC AGGACGCACC TTCAGGAACT ATCCCATGGC CTGGTTCCGC 180

CAGGCTCCAG GAAAGGAGCG TGAGTTTGTA GCAGGTATTA CCTGGGTCGG TGCTAGCACA 240

CTCTATGCAG ACTTCGCGAA GGGCCGATTC ACCATCTCCA GAGACAACGC CAAGAACACG 300

GTGTATCTGC AAATGAACAG CCTGAAACCT GAGGACACGG CCGTTTATAG TTGTGCAGCA 360

GGTCGCGGTA TAGTGGCTGG TAGGATCCCA GCTGAGTATG CCGACTGGGG CCAGGGCACC 420

CAGGTCACCG TCTCCTCAGA ACCCAAGACA CCAAAACCAC AACCAGCGGC CGCACATCAT 480

CATCACCATC ACGGGGCCGC AGAACAAAAA CTCATCTCAG AAGAGGATCT GAATGGGGCC 540

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Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala 20

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Arg Leu Ser Cys Thr Gly Ser Gly Arg Thr 50

Phe Arg Asn Tyr Pro Met Ala Trp Phe Arg 60

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Ala Gly Ile Thr Trp Val Gly Ala Ser Thr 80

Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe 90

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AGACTTCTCCT GTGTAGGCTC CGGTAGCAGA TTCGATAACT ATGCCATGGG CTGGTTCCGC 180

CAGGCTCCAG GGAAGGAGCG TGAATTTGTA GCCGCTATTA GCTGGAGTAG TGGCACTACG 240

CGCTATTTAG ACACCGTGAA GGGCCGATTC ACCATCTCCA GAGACAACGC CAAGAGTACG 300

GTATATCTTC AAATGAACAG CCTGAAACCT GAGGACACGG CCGTTTTATTA CTGTGCAGCT 360

CGATATCAGC CGAGGTACTA CGACTCAGGG GATATGGATG GATATGAGTA TGACAACTGG 420

GGTCAGGGGA CCCAGGTCAC CGTCTCCTCA GAACCCAAGA CACCAAAACC ACAACCAGCG 480

GCCGCACATC ATCATCACCA TCACGGGGCC GCAGAACAAA AACTCATCTC AGAAGAGGAT 540

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Arg Leu Ser Cys Val Gly Ser Gly Ser Arg 50

Phe Asp Asn Tyr Ala Met Gly Trp Phe Arg 60

Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 70

Ala Ala Ile Ser Trp Ser Ser Gly Thr Thr 80

Arg Tyr Leu Asp Thr Val Lys Gly Arg Phe 90

Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr 100

Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 120

Arg Tyr Gln Pro Arg Tyr Tyr Asp Ser Gly 130

Asp Met Asp Gly Tyr Glu Tyr Asp Asn Trp 140

Gly Gln Gly Thr Gln Val Thr Val Ser Ser 150

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TTCCGCCAGG CTCCAGGAAA GGAGCGTGAG TTTGTAGCAG GTATTACCTG GGTCGGTGCT 180

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GCAGCAGGTC GCGGTATAGT GGCTGGTAGG ATCCCAGCTG AGTATGCCGA CTGGGGCCAG 360

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GCTGAGTACT TCGAAATGTC CGTTCGGTTG GCAGAAGCTA TGAAACGATA TGGGCTGAAT 660

ACAAATCACA GAATCGTCGT ATGCAGTGAA AACTCTCTTC AATTCTTTAT GCCGGTGTTG 720

GGCGCGTTAT TTATCGGAGT TGCAGTTGCG CCCGCGAACG ACATTTATAA TGAACGTGAA 780

TTGCTCAACA GTATGGGCAT TTCGCAGCCT ACCGTGGTGT TCGTTTCCAA AAAGGGGTTG 840

CAAAAAATTT TGAACGTGCA AAAAAAGCTC CCAATCATCC AAAAAATTAT TATCATGGAT 900

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CCCGGTTTTA ATGAATACGA TTTTGTGCCA GAGTCCTTCG ATAGGGACAA GACAATTGCA 1020

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CTGAGGAGCC TTCAGGATTA CAAGATTCAA AGTGCGCTGC TGGTGCCAAC CCTATTCTCC 1320

TTCTTCGCCA AAAGCACTCT GATTGACAAA TACGATTTAT CTAATTTACA CGAAATTGCT 1380

TCTGGTGGCG CTCCCCTCTC TAAGGAAGTC GGGGAAGCGG TTGCCAAGAG GTTCCATCTG 1440

CCAGGTATCA GGCAAGGATA TGGGCTCACT GAGACTACAT CAGCTATTCT GATTACACCC 1500

GAGGGGGATG ATAAACCGGG CGCGGTCGGT AAAGTTGTTC CATTTTTTGA AGCGAAGGTT 1560

GTGGATCTGG ATACCGGGAA AACGCTGGGC GTTAATCAAA GAGGCGAACT GTGTGTGAGA 1620

GGTCCTATGA TTATGTCCGG TTATGTAAAC AATCCGGAAG CGACCAACGC CTTGATTGAC 1680

AAGGATGGAT GGCTACATTC TGGAGACATA GCTTACTGGG ACGAAGACGA ACACTTCTTC 1740

ATCGTTGACC GCCTGAAGTC TCTGATTAAG TACAAAGGCT ATCAGGTGGC TCCCGCTGAA 1800

TTGGAATCCA TCTTGCTCCA ACACCCCAAC ATCTTCGACG CAGGTGTCGC AGGTCTTCCC 1860

GACGATGACG CCGGTGAACT TCCCGCCGCC GTTGTTGTTTGGAGCACGG AAAGACGATG 1920

ACGGAAAAAAG AGATCGTGGA TTACGTCGCC AGTCAAGTAA CAACCGCGAA AAAGTTGCGC 1980

GGAGGAGTTG TGTTTGTGGA CGAAGTACCG AAAGGTCTTA CCGGAAAACT CGACGCAAGA 2040

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Ser Leu Arg Leu Ser Cys Thr Gly Ser Gly 30

Arg Thr Phe Arg Asn Tyr Pro Met Ala Trp 40

Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 50

Phe Val Ala Gly Ile Thr Trp Val Gly Ala 60

Ser Thr Leu Tyr Ala Asp Phe Ala Lys Gly 70

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 80

Asn Thr Val Tyr Leu Gln Met Asn Ser Leu 90

Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys 100

Ala Ala Gly Arg Gly Ile Val Ala Gly Arg 110

Ile Pro Ala Glu Tyr Ala Asp Trp Gly Gln 120

Gly Thr Gln Val Thr Val Ser Ser Glu Pro 130

Lys Thr Pro Lys Pro Gln Pro Ala Glu Leu 140

Pro Gly Ala Ala Ala Cys Arg Met Glu Asp 150

Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro 160

Phe Tyr Pro Leu Glu Asp Gly Thr Ala Gly 170

Glu Gln Leu His Lys Ala Met Lys Arg Tyr 180

Ala Leu Val Pro Gly Thr Ile Ala Phe Thr 190

Asp Ala His Ile Glu Val Asp Ile Thr Tyr 200

Ala Glu Tyr Phe Glu Met Ser Val Arg Leu 210

Ala Glu Ala Met Lys Arg Tyr Gly Leu Asn 220

Thr Asn His Arg Ile Val Val Cys Ser Glu 230

Asn Ser Leu Gln Phe Phe Met Pro Val Leu 240

Gly Ala Leu Phe Ile Gly Val Ala Val Ala 250

Pro Ala Asn Asp Ile Tyr Asn Glu Arg Glu 260

Leu Leu Asn Ser Met Gly Ile Ser Gln Pro 270

Thr Val Val Phe Val Ser Lys Lys Gly Leu 280

Gln Lys Ile Leu Asn Val Gln Lys Lys Leu 290

Pro Ile Ile Gln Lys Ile Ile Ile Met Asp 300

Ser Lys Thr Asp Tyr Gln Gly Phe Gln Ser 310

Met Tyr Thr Phe Val Thr Ser His Leu Pro 320

Pro Gly Phe Asn Glu Tyr Asp Phe Val Pro 330

Glu Ser Phe Asp Arg Asp Lys Thr Ile Ala 340

Leu Ile Met Asn Ser Ser Gly Ser Thr Gly 350

Leu Pro Lys Gly Val Ala Leu Pro His Arg 360

Thr Ala Cys Val Arg Phe Ser His Ala Arg 370

Asp Pro Ile Phe Gly Asn Gln Ile Ile Pro 380

Asp Thr Ala Ile Leu Ser Val Val Pro Phe 390

His His Gly Phe Gly Met Phe Thr Thr Leu 400

Gly Tyr Leu Ile Cys Gly Phe Arg Val Val 410

Leu Met Tyr Arg Phe Glu Glu Glu Leu Phe 420

Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln 430

Ser Ala Leu Leu Val Pro Thr Leu Phe Ser 440

Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys 450

Tyr Asp Leu Ser Asn Leu His Glu Ile Ala 460

Ser Gly Gly Ala Pro Leu Ser Lys Glu Val 470

Gly Glu Ala Val Ala Lys Arg Phe His Leu 480

Pro Gly Ile Arg Gln Gly Tyr Gly Leu Thr 490

Glu Thr Thr Ser Ala Ile Leu Ile Thr Pro 500

Glu Gly Asp Asp Lys Pro Gly Ala Val Gly 510

Lys Val Val Pro Phe Phe Glu Ala Lys Val 520

Val Asp Leu Asp Thr Gly Lys Thr Leu Gly 530

Val Asn Gln Arg Gly Glu Leu Cys Val Arg 540

Gly Pro Met Ile Met Ser Gly Tyr Val Asn 550

Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp 560

Lys Asp Gly Trp Leu His Ser Gly Asp Ile 570

Ala Tyr Trp Asp Glu Asp Glu His Phe Phe 580

Ile Val Asp Arg Leu Lys Ser Leu Ile Lys 590

Tyr Lys Gly Tyr Gln Val Ala Pro Ala Glu 600

Leu Glu Ser Ile Leu Leu Gln His Pro Asn 610

Ile Phe Asp Ala Gly Val Ala Gly Leu Pro 620

Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala 630

Val Val Leu Glu His Gly Lys Thr Met 640

Thr Glu Lys Glu Ile Val Asp Tyr Val Ala 650

Ser Gln Val Thr Thr Ala Lys Lys Leu Arg 660

Gly Gly Val Val Phe Val Asp Glu Val Pro 670

Lys Gly Leu Thr Gly Lys Leu Asp Ala Arg 680

Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys 690

Lys Gly Gly Lys 694

Claims (6)

1. a detection method of soluble CD38 concentration, is characterized in that, comprises the steps: S1. Use single domain antibody 551 as a capture antibody, coated on the bottom of the ELISA plate to capture CD38 in the sample solution; S2. Using the fusion protein of single domain antibody 1053 and luciferase as the detection antibody, which specifically binds to CD38; S3. Detecting the luciferase-catalyzed luciferin luminescence signal to evaluate the CD38 concentration. 2. The method for detecting the concentration of soluble CD38 according to claim 1, wherein the luciferase is a firefly luciferase mutant Fluc2. 3. The method for detecting the concentration of soluble CD38 according to claim 1, wherein the amino acid sequence of the single domain antibody 551 is shown in sequence 4 in the sequence listing, and its coding gene is shown in sequence 3 in the sequence listing. 4. The method for detecting the concentration of soluble CD38 according to claim 1, wherein the amino acid sequence of the single domain antibody 1053 is shown in sequence 2 in the sequence listing, and its coding gene is shown in sequence 1 in the sequence listing. 5. The method for detecting the concentration of soluble CD38 according to claim 1, wherein the amino acid sequence of the detection antibody is shown in sequence 6 in the sequence listing, and its coding gene is shown in sequence 5 in the sequence listing. 6. the detection method of soluble CD38 concentration according to claim 1, is characterized in that, the expression vector of described detection antibody, construction process comprises the steps: S201. Amplify the gene sequence of the CD38 single domain antibody 1053 by PCR, digest the pRHSUL2 vector and the 1053 gene sequence with restriction enzymes KpnI and SacI, and connect the two fragments with T4 DNA ligase to construct pRHSUL2-1053; S202. Digest the pRHSUL2-1053 vector with restriction endonucleases SacI and PstI, anneal the two DNA single strands containing SacI and PstI cohesive ends and 2×G ₄ S sequences, and use T4 DNA ligase to 2×G ₄ Insert S into pRHSUL2-1053 to construct pRHSUL2-1053-G ₄ S; S203. PCR amplifies the gene sequence of the firefly luciferase mutant Fluc2, cuts the pRHSUL2-1053-G ₄ S and Fluc2 gene sequences with restriction endonucleases PstI and HindIII, and connects the two fragments with T4 DNA ligase, Construct pRHSUL2-1053-G ₄ S-Fluc2, which is the expression vector of the detection antibody.