AU2022283733B2 - Helicobacter pylori detection kit and detection method thereof - Google Patents

Abstract

The present invention discloses a Helicobacter Pylori (HP) detection kit and a detection method thereof, falling within the field of immunodetection. Specifically, the present invention discloses a specific HP-binding antibody, a kit comprising the same and a detection method thereof. The specific HP-binding of the present invention can achieve accurate and sensitive detection of HP. The method is simple and fast, and easy to popularization and application. 18

Description

AustralianPatents Australian PatentsAct Act1990 1990

ORIGINAL COMPLETE ORIGINAL SPECIFICATION COMPLETE SPECIFICATION STANDARD PATENT

Invention Title Invention Title

Helicobacter pylori detection kit and detection method thereof

The following statement is a full description of this invention, including the best method of performing it known to me/us:

1

Description

FIELD FIELD OF OF TECHNOLOGY TECHNOLOGY The present invention falls within the field of biological immunodetection, and relates to a helicobacterpylori(HP) detection kit and a detection method thereof.

BACKGROUND BACKGROUND Helicobacterpylori(HP) is a kind of spiral Gram-negative pathogenic bacterium from human

to infect the lower part of the gastric mucous layer. HP has smooth bacterial cells and are easy to be

adhered on the mucosa of sinuses ventriculi and gastric body near pylorus. Because HP is located at the deep layer of gastric mucus, HP is not in direct contact with gastric acid. HP can lead to chronic

gastritis peptic ulcer and non-ulcer dyspepsia. HP is clinically divided into two types: type I and

type II; type I strain has very strong pathogenicity and thus, is easy to cause gastric diseases, while type II generally has no clinical symptoms after infection.

A large number of clinical data has proved that HP infection is related to chronic gastritis,

peptic ulcer, primary gastric B-cell lymphoma and gastric cancer. Therefore, it seems crucial to make accurate and effective diagnosis on the HP infection, and then to clear away HP from the

infected patients.

Currently, HP detection methods can be summarized into two major categories: an invasive method and a non-invasive method. The invasive method is suitable for the subject capable of

receiving gastrointestinal endoscopy; HP is detected by collecting gastric mucosa tissues, for

example, by rapid urease test (RUT), pathological staining detection method, bacterial cultivation andthe and thelike. like.

On the other hand, HP diagnosis mainly relies on 13C urea breath test clinically, namely, a

non-invasive method. However, the test can be only performed at least 4-8 weeks later after the drug therapy; moreover, it is forbidden for children and pregnant women due to radiation.

Furthermore, HP can be also detected effectively by PCR detection on the urease gene and

toxin-related protein gene A of HP in gastric juice or mucosa. However, the detection requires a longer period and complex operation.

The detection of HP antibody in blood serum by immunohistochemistry is a kind of optional non-invasive method, of which enzyme linked immunosorbent assay and Western Blotting are

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relatively common. However, low sensitivity of the antibody used in the method is the problem to be solved urgently.

SUMMARY Based on the problem existing in the prior art, the objective of the present invention is to study

and develop a specific single domain antibody capable of identifying HP. Inventors of the present

invention keep trying to isolate a variable domain of heavy chain of heavy-chain antibody (VHH) directed to HP from a phage-display antibody library. Specifically, the VHHs have sequences as

shownininTable shown Table11 below. below. Table 1: Complete sequence of the single domain antibody and CDR thereof Nameof VHH amino acid sequence (the CDR regions are bold and VHH inclined letters) QVQLVGGGESGGTLSVGGSLRFCAASASGPYGDVM WFNRQADRTPKSVGSKGIHVHGAGNYRTYVAARFDI TSTRYANRNLTQMNSLNAKPEDVYYCATTRGYRSYC GSQDWNVRYWGGIQTVVQSQ ELVVQEGTVQPPGRDFSSLLSACGFVSTMNMSYWV RQPAGVKCGGYTRDIGGSGSNDYIYDASKVGRFTIV AYC09 DNKNVNTSYLTAQMELKPDTVYAYNCADFWPYIWG DWGGQSTQKNAS QVQLSGVEGGSVASLGGCRLSAGASSRAFPYGGMW FRGRSFIRFTEFVAAITGWASSTYRDYGKGQIRFTVYI AYC32 SRDISNAKMASNTDKLQLEPAACAYDRPWEDGGSG YGYSQNRWGQQGTVTSVS QVQLVEGGLVQPSGGSLSCRLIFVASGFSDMYGLWA APGRQAKTVASPVAINRTGGTKNYSNGIVRFRETVSD AYC58 NRDVTKNVNASYDLKYDYTAMYACYQDGLNTSTS VNYAYGWGRISQVVTS

Based on the above objective of the present invention, the present invention specifically an-HP

single domain antibody AYC03, AYC09, AYC32 and AYC58 firstly. Amino acid sequences of the regions CDR1, CDR2 and CDR3 on the heavy chain of the antibody are respectively shown in the

boldand bold andinclined inclined lettersin in letters thethe table table above. above.

The present invention provides an anti-HP antibody (e.g., conventional quadruplex-stranded

antibody, domain antibody, single-chain antibody, VHH, bispecific antibody, or poly-specific antibody, or the like) or an antigen-binding fragment thereof, including the anti-HP heavy-chain

single domain antibody in the table above or three CDRs: CDR1, CDR2 and CDR3 thereof.

In the other aspect, the present invention relates to a nucleic acid molecule encoding the

anti-HP antibody and an expression vector and a host cell containing the nucleic acid molecule.

The present invention further relates to a method for preparing an anti-HP antibody.

The present invention further relates to an application of the anti-HP antibody, in particular to

use and method in diagnosis of an anti-HP-related disease.

Further, the present invention further provides a variant of a HP-directed antibody, where the

variant is a humanized variant or a variant having an identity of (for example, at least 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 5 %, 96%, 9 7 %, 98%, 99%, 99.5% and 99.9%).

Exemplarily, the antibody of the present invention is selected from Ig, Ig NAR, Fab fragment,

Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bis-scFv, (scFv)2, micro-antibody, double-stranded antibody, triple-stranded antibody, quadruplex-stranded antibody, Fv protein with a

stable disulfide bond and single domain antibody, bispecific antibody or trispecific antibody of

camel, preferably, the VHH named AYC03, AYC09, AYC32 and AYC58 or a humanized variant

thereof. thereof.

The present invention further provides a fusion protein, including the antibody and the variant thereof or the antigen-binding fragment thereof of the present invention, for example, the fusion

protein contains a tag sequence (e.g., Poly-His) or an IgG1-Fc protein sequence.

The present invention further provides a polynucleotide of an isolated expression antigen or an

antigen-binding fragment thereof, where the polynucleotide is capable of expressing the antibody

and the variant thereof or the antigen-binding fragment thereof in the present invention; or the

polynucleotide is capable of expressing the fusion protection of the present invention.

The present invention further provides a vector, including the polynucleotide of the present

invention, preferably, a plasmid vector.

The present invention further provides a host cell, including the polynucleotide or the vector of the present invention, preferably, where the host cell is eukaryocyte.

The present invention further provides a method for detecting whether HP is contained in a sample, including a step of contacting the antibody and the variant thereof or the antigen-binding

fragment thereof, or the fusion protection in the present invention with the sample, preferably, the detection may be for the purpose of diagnosis, or not for the purpose of diagnosis.

The present invention further provides a detection product, including the antibody and the variant thereof or the antigen-binding fragment thereof in the present invention; preferably, the

product is selected from one or more of a detection reagent, a kit, a chip or a test paper.

A single domain antibody is applied in this present invention and thus, has a small molecular weight. Therefore, it can be considered that to reduce the possibility of detecting false-negative HP

as much as possible, the variable domain of heavy chain of heavy-chain antibodies (directed to

different epitopes) of the present invention are linked via a connexin or linked in series directly to form a bispecific antibody.

All the terms used herein have common meanings in the art, unless otherwise indicated or

defined; the meanings will be understood by a person skilled in the art. For example, unless otherwise specified, the term "antibody" or "immunoglobulin" capable of

being exchanged may refer to a heavy chain antibody, a variable domain of heavy chain of

heavy-chain antibody or conventional quadruplex-stranded antibody, for example, may refer to a full-length antibody, a single chain thereof, single-chain antibody (scFv) and structural domain or

fragment thereof (for example, antigen binding domain or fragment, respectively for example, VHH

domain or VH/VL domain), or may refer to a bispecific antibody or poly-specific antibody. Moreover, the term "sequence" used herein (for example, in the use of "immunoglobulin", "antibody", "variable domain of heavy chain of heavy-chain antibody", "VHH", "protein" or

"binding protein", and the like), generally includes related amino acid sequences, and further includes nucleotide sequence or nucleotide sequence encoding the sequence, which may be

obviously determined by a person skilled in the art, unless otherwise specified herein.

An example of the domain antibody of the present invention may be "VHH"of single variable domains VH and VL (VH domain and VL domain) or Camelidae. "VHH"is also called a variable

domain of heavy chain of heavy-chain antibody, a VHH domain, a VHH antibody fragment and a

VHH antibody. VHH specifically binds to an epitope without other antigen binding domains. Terms "variable domain of heavy chain of heavy-chain antibody", "VHH structural domain", "VHH",

"VHH domain", "VHH antibody fragment", and "VHH antibody" in the context of the present

invention may be exchanged. The inventor of the present invention performs uncertainty screening by using a large number

of HP-directed holoproteins as antigens for the first time to obtain an antibody capable of binding to

HP holoproteins, for example, VHH. Optionally, the binding is specific binding.

In this present disclosure, the binding may be "specific binding". As understood in the art, a person skilled in the art may know various test methods or means capable of being used for testing binding or specific binding very well. Methods for determining an equilibrium association constant or an equilibrium dissociation constant are known in the art. Some cross reactions or background bindings are possible to occur in interactions between lots of proteins, but which will not damage the "specific" binding of antibodies or active fragments thereof. The "specific binding", for example, may refer to the binding of the anti-HP antibody or antigen-binding fragment thereof in this present disclosure to HP or protein thereof, and the binding affinity is higher than background binding.

The antibody or antibody fragment herein includes but not limited to polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single-chain variable fragments (scFv),

, single domain antibodies (for example, VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression library, anti-idiotypic antibodies, epitope binding fragments or any combination of the above on condition that the above antibodies have binding properties similar to those of the antibody in the present disclosure, preferably, includes the corresponding CDRs, or VH and VL regions described herein. Tiny antibodies and polyvalent antibodies, such as bivalent antibodies, trivalent antibodies, tetravalent antibodies and pentavalent antibodies may be also used in the present invention.

As used herein, the "antibody" generally refers to a protein composed of one or more polypeptides basically encoded by immunoglobulin genes or immunoglobulin gene fragments. When the term "antibody" is used, it may be also considered to represent an "antibody fragment". Optionally, the antibody or antibody fragment may be chemically conjugated with other proteins or fusion proteins thereof or expressed as a fusion protein carrying other proteins. In some embodiments, the antibody or antigen-binding fragment of the present invention is contained in a poly-specific antibody, for example, a bispecific antibody. Such a poly-specific antibody may be produced by a method known in the art, for example, by crosslinking two or more same or different types of antibodies or antigen-binding fragments (e.g., scFv). A humanized antibody may be prepared by a method known in the art; the humanized antibody contains one or more CDRs of the antibody or antibody fragment of the present invention or contains one or more CDRs derived from the antibody or antibody fragment, and contains a humanized FR region. For example, a monoclonal antibody may be generally humanized by four steps of: (1) determining nucleotides of the light-chain and heavy-chain variable domains of an 5

antibody and predicted amino acid sequences; (2) designing a humanized antibody, namely,

determining what kind of antibody framework region is used in the humanization process; (3)

performing the humanization method/technology; and (4) transfection and expression of the humanized antibody.

The term humanized antibody means that at least a portion of framework region of

immunoglobulin and a portion of optional CDR region or other regions participating in binding is derived from or adjusted as a human immunoglobulin sequence. The humanization, chimeric

process or partial humanized forms of mouse monoclonal antibodies are achieved by, for example,

a recombination DNA technique. The CDR region of a non-human antibody is linked to a human constant region by the recombinant DNA technique, which may produce humanized mouse

antibodies. Optionally, the monoclonal antibody used in the present invention may be a human

monoclonal antibody. A human antibody may be obtained by, for example, a phage display

technique.

As used herein, the humanized antibody further refers to a form of a non-human (for example, murine, camel, yamma and shark) antibody, which is a specific chimeric immunoglobulin

containing the minimal sequence derived from a non-human immunoglobulin, immunoglobulin

light chain or fragments thereof (for example, Fv, Fab, Fab', F(ab')2 or other antigen binding

subsequences of an antibody, for example, vHH.

As used herein, the human or humanized antibody or antibody fragment refers to the antibody having amino acid sequences corresponding to the amino acid sequences of antibodies produced by

human, and may be prepared by any technology for preparing antibodies known in the art. A human

antibody or fragment thereof may be chosen by a competitive binding experiment or other ways, thus determining whether the human antibody or fragment thereof has epitope binding specificity the same as that of the specific mouse antibody.

Variable region and complementary determining region (CDR)

The variable region of an antibody refers to a separate variable region of light chain of an

antibody or a separate variable region of heavy chain of an antibody or a combination of the two.

The variable region of heavy chain and variable region of light chain respectively consist of four framework regions (FR) connected via three complementary determining regions (CDR); the three

CDRs are also called hypervariable regions. CDRs in each chain are kept together closely via FR,

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and conducive to the formation of antigen binding sites of an antibody with CDRs from other chains. chains.

CDR is mainly responsible for binding epitopes. There are many methods capable of being used for determining borders of CDR amino acid sequences, for example, Kabat, et al., Sequences

of Proteins of Immunological Interest (the 5th edition, 1991, National Institutes of Health, Bethesda

Md, "Kabat" numbering scheme); Al-Lazikani B, Lesk AM, Chothia C. Standard conformations for the canonical structures of immunoglobulins. J. Mol. Biol. 1997; 273:927-48 ("Chothia" numbering

scheme); Lefranc, et al., ("IMGT unique numbering for immunoglobulin and T cell receptor

variable domain sand Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme);or North, B, Lehmann A, Dunbrack R. A new clustering of antibody

CDR loop conformations: J. Mol. Biol. (2011), 406(2): 228-256. Besides the above methods, the

alternative methods further include new solutions developed with the development of bioinformatics. Kabat is the most common method, but CDR may refer to CDR defined by one or

more methods, or by a combination of these methods.

DESCRIPTION OF DESCRIPTION OF THE THE EMBODIMENTS EMBODIMENTS To describe the objective, technical solutions and advantages of the present invention more

clearly, the embodiments of the present application will be further described in detail by reference

to the accompanying drawings.

Example 1: preparation of HP antigen

Cultured HP was scraped and dissolved into a buffer solution, and centrifuged for 5 min at 10000 rpm; then precipitate was taken and resuspended by normal saline, and added with 0.5%

formaldehyde based on the bacterial suspension, and the treated precipitate was subjected to

standing for 24 h at 37C and then washed for three times with normal saline, and the concentration of the bacteria solution was adjusted to 1x108 cfu/ml to obtain an inactivated HP bacterial

suspension, and the inactivated HP bacterial suspension was ultrasonically lysed to obtain the HP

holoprotein. The HP holoprotein solution was added with an EDTA buffer solution and treated in a 95°C

water bath for 10 min to obtain a HP antigen protein solution; the HP holoprotein solution was adjusted to a final concentration of 0.2 mg/mL, then cryopreserved.

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Example 2: animal immunization Alpaca was injected with a HP antigen protein on the neck and back subcutaneously and

intramuscularly in a multipoint way to form multiple enclosed masses, and immunization was

verified correct. verified correct. First immunization: 2.5 ml antigen and Freund's complete adjuvant were mixed by 1:1, and

emulsified for injection;

second immunization: 3 weeks later after the first immunization, 1 ml antigen and Freund's complete adjuvant were mixed by 1:1, and emulsified for injection;

third immunization: 3 weeks later after the second immunization, 1 ml antigen and Freund's

complete adjuvant were mixed by 1:1, and emulsified for injection; and four immunization: 3 weeks later after the third immunization, 1 ml antigen and Freund's

complete adjuvant were mixed by 1:1, and emulsified for injection;

One week later after the four times of immunization, 2 ml peripheral blood was collected to separate blood serum. The HP holoprotein was coated into an ELISA 96-well plate to determine the

valence of antibody in the blood serum via ELISA. The ELISA results show that after four times of

immunization, the alpaca has a serum valence greater than 1:32000 to accord with the library-building standards.

Example 3: establishment of the natural antibody library with high storage capacity After four times of immunization, the alpaca has a valence serum greater than 1:32000, which

indicates that there exist anti-HP high affinity antibodies in blood serum. Therefore, the

phage-display immune antibody library was constructed according to the following steps:

1. Peripheral blood of alpaca after being immunized for the fourth time was collected to isolate

lymphocyte PBMC. Specifically, the peripheral blood was diluted with a RPMI-1640 culture solution containing 10% heparin by 1:1, and then added to a centrifugal tube with a lymphocyte

separation solution (the volume ratio of the solution for diluting venous blood to the lymphocyte

separation solution was 2:1), and then 2000 g were centrifuged for 20 min. The mononuclear cell layer was absorbed and washed for twice with a PBS buffer solution.

2. 2x107 PBMCs were taken to extract total RNA by a RNA extraction kit. Specifically, 4 ml Trizol reagent was added to pipette the cells for lysis. The cells were incubated for 5 min at room

temperature, and transferred to a DEPC-treated EP tube, and added with chloroform in 1/5 volume,

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2022283733 08 2022

and violently oscillated, and incubated for 3 min at room temperature. 10,000 g were centrifuged for 15 min at 4C; the upper layer of aqueous phase was absorbed to a new centrifugal tube, and Dec added with isopropanol in 1/2 volume, and then treated in an ice bath for 10 min. 12,000 g were centrifuged for 10 min at 4C, and then supernatant was discarded, and 1 ml 75% ethanol was added to wash the precipitate. 7,500 g were centrifuged for 5 min at 4C, and then supernatant was discarded, the precipitate was dried at room temperature and dissolved into RNase-free water or sedimented into absolute ethanol, and then cryopreserved at -80°C.

3. RNA was taken to prepare cDNA by a RT-PCR reverse transcription kit. The total RNA was treated by RNase-free DNase I first to remove the residual genomic DNA. 2 pg treated RNA samples and Oligo (dT)15 (500 pg/ml)1pl were taken, and refilled with DEPC water to 12 pl, and then heated for 10 min at 70°C. After being taken out, the samples were immediately placed into an ice bath, and successively added with 5xBuffer 5pl, dNTP (10 mmol/L) 5pl, RNA enzyme inhibitor lpland MMLV reverse transcriptase 1pl, and then refilled with DEPC water to 25pl, and subjected to heat preservation for 60 min at 42C for reverse transcriptional reaction.

4. Sequences of the variable region of heavy chain of IgG2 and IgG3 (heavy-chain variable region VHH of a nanobody) were obtained by nested PCR substeps, and the experimental procedures were as follows: 1) a pair of specific nested outside primer were designed for the first round of PCR amplification with cDNA as a template; the amplified region was the Leader-CH2 region of a heavy chain antibody gene of alpaca; the amplified product had a size of about 700 bp, and the PCR product was recovered by DNA gel electrophoresis and gel cutting.

PCR parameters: the primers were denatured for 3 min at 94C, and denatured for 30 sec. at 94°C, for 30 sec. at 61°C, and for 1 min at 72C, and then subjected to PCR with 30 cycles, and finally elongated for 10 min at 72C. At the end of the reaction, 5 1 reaction product was taken and subjected to 1% agarose gel electrophoresis for analysis.

2) Nested inside primers were designed for the second round of PCR amplification with the first round of PCR product as a template; the amplified region was the VHH fragment of the variable region of heavy chain antibody of alpaca; and the second round of PCR product was purified and recovered by a PCR product purification kit.

The PCR product had the following recovery steps:

(1) the PCR product was subjected to 1.5% agarose gel electrophoresis to cut off the target DNA fragment from the agarose gel, and then the gel was put to a 1.5 ml centrifugal tube;

(2) a gel lysis buffer was added to dissolve the gel fully;

(3) the full-dissolving solution was absorbed into a recovery column;

(4) 12,000 g were centrifuged for 1 min, and waste solution was discarded;

(5) 500 pl neutralizing solution was added; 12,000 g were centrifuged for 1 min, and waste solution was discarded;

(6) 700 pl washing solution was added; 12,000 g were centrifuged for 1 min, and waste solution was discarded;

(7) 12,000 g were centrifuged for 2 min, and waste solution was discarded; the recovery column was transferred onto a receiving tube, and dried for 5 min at room temperature; and

(8) 30pl deionized water was added, and 12,000 g were centrifuged for 1 min, and the DNA fragment was eluted and cryopreserved at -20°C.

5. Sequences of the variable region of heavy chain were inserted into a digested linearized phasmid vector in a digestion and linkup way to obtain a recombinant vector; after being purified

and recovered, the recombinant vector was transformed into competent cells SS320 (containing a

helper phage M13K07); the transformed bacterial solution was resuspended by a medium and activated for 1 h; a small amount of bacterial solution was taken and diluted by 10-fold gradients,

and coated on a LB/Carb50 culture plate and cultured over the night at 37C and used for the

calculation of storage capacity; the remaining bacterial solution was cultured over the night on a liquid medium to collect supernatant; the phage was precipitated from the supernatant and then the

precipitate was resuspended by a PBT solution, thus obtaining the phage-display immune antibody

library (cryopreserved at -80°C). 6. Statistics was performed on the number of clones on the LB/Carb50 plate to calculate the

storage capacity as follows: the storage capacity of the alpaca antibody library was 2.16x109, which

completely satisfied the demands for screening antibodies.

Example 4: enrichment of the anti-HP antibody library

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1. 5 pg/mL HP holoprotein was added to a 96-well plate (100 pl/well), and coated over the night at 4C; NEB5aF' competent Escherichia coli was subjected to streaking growth, and cultured

over the night in a 37C incubator.

2. NEB5aF' monoclonal antibodies were picked from the 2YT/Tet10 plate cultured over the night on the next day, and added to 3 ml 2YT/Tet0 liquid medium, and subjected to oscillation

growth at 37C to OD600=0.8.

3. Meanwhile, the antigen supernatant on the 96-well plate was removed and 200 pL 1% BSA was added to per well for blocking, and 200 pL 1% BSA was added to a blank well as a negative

control well, and then placed into a 3D rotary oscillator for 2 h at room temperature; afterwards,

supernatant in the protein well and blank well was removed, and washed with 200 pL cleaning agent PT, and each well was added with 100 pL phage-display antibody library and placed into the

3D rotary oscillator for 2 h at room temperature; supernatant in the protein well and blank well was

removed, and washed with 200 pL PT; and 100 pL 100 mM HCl was added to the well and placed for 5 min at room temperature, and then supernatant was sucked out and added to a 1.5 ml

centrifugal tube, and neutralized by IM Tris-HCl.

4. The mixed solution obtained in the step 3 was added to a centrifugal tube containing 1 mL NEB5aF' bacterium and cultured for 1 h on a table concentrator at 37C; then 20 pL culture

solution in the centrifugal tube was taken and diluted by suitable folds, and coated on the LB/Carb5 o

culture plate, and then the culture plate was placed into a 37C biochemical incubator over the night, the incubated solution was used for the calculation of titer and degree of enrichment on the

next day; the remaining culture solution was added with 1 pL helper phage M13K07 (a final

concentration of 10 10/mL), and cultured for 1 h on a table concentrator at 37C; then the above culture solution was transferred to 35 mL 2YT/Carbo/Kan2 5 culture solution and placed into the

table concentrator, and cultured over the night at 37C; then the phage was collected to form each

round of antibody library. 5. The above operating steps were repeated for 3-5 rounds until the appearance of phage

enrichment. The colony forming unit (CFU) of the antigen binding well on the LB/Carb5 o culture

plate is above 10 folds of that of the negative control well, the enrichment is considered successful. In this experiment, after the third round of screening, the CFU of the antigen binding well is 100

folds of that of the negative control well, indicating enrichment success.

Example 5:

1. 400 pL 2YT/Carb5 o/Kan2 5/M3K07 medium was added to per well of the 96-deep-well plate; monoclonal antibodies were picked from the obtained LB/Carb 5 o culture plate enriched, and

transferred into the 96-deep-well plate; then the 96-deep-well plate was put on a table concentrator

over the night at 200 rpm and 37C, and then centrifuged on the next day; supernatant was namely the phage produced by each monoclonal antibody.

2. Meanwhile, the HP holoprotein was diluted to 5 tg/mL, and added to an ELISA 96-well

plate by 50 pL/well, and placed into a 4C refrigerator over the night. 3. The ELISA plate was made upside down on the next day to remove supernatant, and then

100 pL I%BSA was added per well for blocking; 100 pL I%BSA was added to the blank well as a

negative control well, and then incubation was performed for 1 h at room temperature. At the end of the blocking, the antigen well and negative control well were washed with a PT solution, and then

added with 50 pL supernatant of the 96-deep-well plate for incubation for 2 h at room temperature;

supernatant obtained from each monoclonal antibody was receptively added to an antigen well and a negative control well.

4. At the end of the binding, the ELISA plate was washed with a PT cleaning agent and added

with 50 pL HRP-labeled M13 antibody for incubation for 1 h at room temperature; then after being washed by the PT solution and PBS solution, the ELISA plate was added with 50 pL TMB for

incubation for 5 min at room temperature, and then added with 50 pL IM phosphoric acid to

terminate the reaction; the absorbance value at 450 nm was measured by ELIASA. If the OD value is greater than 4.0, the ELIASA reading shows overflow.

5. The monoclonal antibodies whose OD value of the antigen well was greater than 0.5 and

OD value of the negative control well was less than 0.2 were recognized as positive clones with higher affinity; there were 28 in total; the monoclonal antibodies whose OD value was greater than

4.0 were selected for sequencing. The results are shown in Table 2 below.

Table 2: Positive clone sequencing and OD value Nameof Name of Amino acid sequence of VHH OD value OD value VHH QVQLVGGGESGGTLSVGG Overflow SLRFCAASASGPYGDVM SLRFCAASASGPYGDVM WFNRQADRTPKSVGSKGI HVHGAGNYRTYVAARFDI AYC03 TSTRYANRNLTQMNSLNA KPEDVYYCATTRGYRSYC KPEDVYYCATTRGYRSYC GSQDWNVRYWGGIQTVV QSQ QSQ

ELVVQEGTVQPPGRDFSSL Overflow Overflow LSACGFVSTMNMSYWVR QPAGVKCGGYTRDIGGSG AYC09 SNDYIYDASKVGRFTIVD NKNVNTSYLTAQMELKPD TVYAYNCADFWPYIWGD WGGQSTQKNAS QVQLSGVEGGSVASLGGC 3.86-Overflow 3.86-Overflow RLSAGASSRAFPYGGMWF RGRSFIRFTEFVAAITGWA AYC32 AYC32 SSTYRDYGKGQIRFTVYIS RDISNAKMASNTDKLQLE PAACAYDRPWEDGGSGY GYSQNRWGQQGTVTSVS QVQLVEGGLVQPSGGSLS 3.52-Overflow CRLIFVASGFSDMYGLWA APGRQAKTVASPVAINRTG AYC58 AYC58 GTKNYSNGIVRFRETVSD GTKNYSNGIVRFRETVSD NRDVTKNVNASYDLKYD NRDVTKNVNASYDLKYD YTAMYACYQDGLNTSTSV NYAYGWGRISQVVTS

Example 6: eukaryotic expression (VHH-Fc fusion protein) and affinity identification of a nanobody Four sequences with overflowing OD values screened in Example 5 were subjected to eukaryotic expression to study the affinity thereof with the HP holoprotein. 1. VHH fragments of these sequences were amplified by PCR, and inserted into an eukaryotic expression vector pFcIG containing partial fragments (hinge+CH2+CH3) of human IgG1 by a homologous recombination or digestion and linkup way, and then the vector was electrically transferred into Escherichia coli trans5a host bacterium to be screened by ampicillin, thus sequencing the monoclonal antibodies to obtain the correct recombinant plasmid; the host bacterium containing the recombinant plasmid was subjected to enlarged cultivation, and then a de-endotoxin kit was used to obtain a sterile endotoxin-free plasmid. 2. HEK293F cells were cultured by a serum-free medium, and the recombinant expression plasmid was transferred into the HEK293F cells for expression by a polyplus suspension cell transfection reagent. 24 h and 72 h later after the transfection, the materials were supplemented, and supernatant was collected on the 5th day; the anti-keratin antibody was isolated and purified by a

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Protein A agarose purification resin, replaced and preserved to a solution of 50 Mm Tris-HCl and 200 mM NaCl (PH=8). By calculation, it can be seen that the VHH-FC fusion proteins of the four

sequences with overflowing OD values have a yield of 8-52 mg/L; SDS-PAGE electrophoresis

identification: the VHH-FC bands of the four sequences have normal size and the purity is greater

than 95%. 3. The ELISA method was used to determine the affinity of the VHH-FC fusion proteins of the

four sequences with the HP holoprotein; 1) the HP holoprotein was added to an ELISA 96-well plate with 100 ng/well, and coated 4C over the night; 2) the VHH-FC fusion proteins were diluted

to different concentrations (0.01-10 pg/ml) and subjected to ELISA reaction with the antigen

holoprotein, and then the absorbance values at 450 nm were measured by ELISA. The results are shown in Table 3. The experimental results indicate that the ELISA EC50 of the VHH-FC fusion

proteins of the four sequences with the HP holoprotein is less than 100pg/mL (1IpM).

Table 3 Binding force (ELISA EC50) between the single domain antibody-Fc and the HP holoprotein Nameof Binding force EC50 with VHH human HP holoprotein (ptg/mL) AYC03 AYC03 0.15 0.15

AYC09 0.20 AYC32 AYC32 0.75 0.75

AYC58 AYC58 0.47

Example 7: selectivity verification of the antibody HP, Escherichiacoli, Streptococcus lactis, Lactobacillus acidophilus,Lactobacillus rhamnosus

and Bifidobacterium were fixed into an ELISA plate, and skimmed milk was added for blocking

and cultured for 2 h at 37C, and then washed to remove the excessive skimmed milk, and washed for 5 times.

10 pL purified VHH-Fc fusion protein was added, oscillated and mixed well, and then

incubated for 1 h at 37C. The incubated fusion protein was then washed for 5 times repeatedly with 200 pL 0.1% Tween 20, thus removing the non-specifically bound VHH-Fc fusion protein.

The obtained solution was added with 200 pL HRP-anti-Fc diluted by skimmed milk for incubation for 1 h at 37C, and then washed to remove the residual liquid. A color developing agent

was added and the reaction was terminated to measure the absorbance value.

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The results show that the four VHH-Fc fusion proteins only specifically bind to HP.

What is described above are merely preferred examples of the present invention, but are not construed as limiting the present invention. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall fall

within the protection scope of the present invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be

understood to imply the inclusion of a stated integer or step or group of integers or steps but not the

exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it),

or to any matter which is known, is not, and should not be taken as an acknowledgment or

admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this

specification relates.

15

Claims (11)

Claims

1. A single variable domain heavy chain (VHH) antibody, wherein the VHH comprises an amino acid sequence of any one of SEQ ID NOs: 1, 5, 9, or 13.

2. A fusion protein, comprising the VHH antibody according to claim 1.

3. The fusion protein according to claim 2, further comprising a tag sequence or an IgG1-Fc 2022283733

protein sequence.

4. A polynucleotide encoding the VHH antibody according to claim 1, or the fusion protein according to claim 2 or 3.

5. A vector comprising the polynucleotide of claim 4.

6. The vector according to claim 5, which is a plasmid vector.

7. A host cell comprising the polynucleotide of claim 4, or the vector of claim 5 or 6.

8. The host cell according to claim 7, which is a eukaryotic cell.

9. A method for detecting Helicobacter pylori (HP) in a sample, the method comprising contacting the sample with the VHH antibody according to claim 1, or the fusion protein according to claim 2 or 3.

10. A kit comprising the VHH antibody according to claim 1, or the fusion protein according to claim 2 or 3, when used to detect Helicobacter pylori (HP) in a sample.

11. The kit according to claim 10, further comprising one or more of a detection reagent, a chip or a test paper.