WO2024061273A1 - Biotin and metabolite blocking agent - Google Patents

Abstract

The present invention relates to a compound represented by formula (I) and blocking agents capable of specifically binding to the compound represented by formula I, biotin, and a metabolite. The biotin blocking agents of the present invention can specifically recognize and bind to biotin and the metabolite thereof in a sample with high sensitivity, eliminate the interference of biotin and the metabolite thereof on a chemiluminescent biotin-streptavidin system, and ensure the accuracy of detection results.

Description

Biotin and metabolite blockers

This application claims the priority of the earlier application submitted to the State Intellectual Property Office of China on September 20, 2022, with patent application number 202211142448.8 and titled "Biotin and Metabolite Blockers". The entirety of this prior application is incorporated by reference into this application.

Technical field

The present invention relates to a blocking agent, in particular to a type of blocking agent that can specifically bind to biotin and metabolites.

Background technique

Biotin (B) is vitamin B7, also known as vitamin H and coenzyme R. It is a sulfur-containing water-soluble vitamin among the B vitamins. Biotin exists in trace amounts in all living cells as a coenzyme of carboxylase and participates in the metabolism of carbohydrates, lipids and proteins. Exogenous biotin is free biotin and its metabolites that exist in the blood after the human body ingests biotin. The daily intake of biotin for the American population is 35 to 70 μg/d, while the recommended appropriate intake for the Chinese population is 5 to 50 mg/d. The population's biotin intake has increased significantly in the past 30 years. This is because biotin is widely used in health care fields such as beauty, hairdressing, manicure, weight control, and pregnancy nutrition, as well as in genetic diseases, biotin deficiency, multiple Clinical treatment of sclerosis, hyperthyroidism, type 2 diabetes, vitamin D poisoning, etc. From 1986 to 2000, the proportion of the U.S. population taking biotin-containing supplements increased from 17% to 33%, with doses usually around 100 μg/d and up to 10,000 μg/d.

The biotin-avidin system (BAS) is an amplification labeling technology that utilizes the property that one molecule of avidin can bind to four molecules of biotin. Streptavidin is a protein with similar biological properties to avidin. Since its non-specific binding to solid phase materials is much less than that of avidin, biotin-streptavidin is often used in practical applications. Synthetic system. BAS-based chemiluminescence technology (chemiluminescence im-munoassay, CLIA) combines the specificity of antigen-antibody reaction, the sensitivity of chemiluminescence reaction and the cascade amplification effect of BAS. It has higher sensitivity and stability, and the detection linear range is wider. It is widely used in the detection of tumor markers, infectious markers, myocardial injury markers, various infectious diseases, hormones, drugs and nucleic acids. BAS-based chemiluminescence determines the analyte through a measurable light signal. The measurable light signal is generated by the biotin label-substance to be measured-luminescent substance label-streptavidin magnetic particle complex, and the amount of this complex is proportional to the light signal. The mechanism of interference of exogenous biotin on BAS-based chemiluminescence is that exogenous biotin and reagent-derived biotin competitively bind to streptavidin-coated microparticles (M). When the exogenous biotin reaches a certain amount, the total amount of biotin in the reaction system exceeds the binding capacity of the streptavidin magnetic particles, resulting in less luminescent substances being captured and the light signal weakening, causing interference.

In addition to biotin, exogenous biotin also includes biotin metabolites. There are two main pathways for biotin catabolism: 1) oxidation of the valeric acid side chain, resulting in bisnorbiotin (Bisnorbiotin) and tetrabisnorbiotin. Formation of biotin and related metabolites as a result of fatty acid oxidation. 2) Oxidation of heterocyclic sulfur atoms leads to the formation of biotin sulfoxide, biotin disulfoxide and biotin sulfone. According to literature reports, the concentration range of biotin in human serum is 133-329 pmol/L, the concentration range of bisnorbiotin is 21-563 pmol/L, and biotin metabolites account for 50-70% of the total amount of all biotin. Therefore, in addition to the interference of biotin on BAS-based chemiluminescence, the concentration of biotin metabolites is also sufficient to affect the detection accuracy of the chemiluminescence kit. Therefore, it is necessary to find a type of biotin blocker that can eliminate the interference of biotin in the body and ensure the detection accuracy of BAS-based chemiluminescence kits.

Contents of the invention

The present invention relates to a blocker that specifically binds compounds of formula I, biotin and metabolites,

Wherein, R ₁ and R ₃ are the same or different, and are independently selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy)-C ₁ -C ₃ alkyl Base, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl -C(=O)-, C ₁ -C ₃ alkylsulfonyl or -YXQ,

where Y is selected from Absent, _CH2 or C(=O),

X is selected from O;

or (CH ₂ )n, wherein n is an integer from 1 to 20;

or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 0, 1, 2 or 3, and k is an integer from 1 to 30;

Or [(CH ₂ )r-CONH]s-(CH ₂ )t, [(CH ₂ )r-CONH]s-[(CH ₂ )tO]u, where r is an integer from 1 to 6 and t is 0 to 5, and s is an integer from 1 to 5, u is an integer from 1 to 5;

Q is H, OH, C(=O)ORq, _NH2 , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety ; Rq is selected from H, succinimide or other active esters;

R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl-C(=O)-, C ₁ -C ₃ alkylsulfonyl;

And when any one of the three substituents R ₁ , R ₂ and R ₃ is selected from H, the remaining two are not H.

As one of the preferred technical solutions, the compound of formula I combined with the blocker, wherein R ₁ is selected from -YXQ,

R ₃ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} or -YXQ,

where Y is _CH2 or C=O,

X is O;

or (CH ₂ )n and n is an integer from 1 to 20;

or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30;

or [(CH ₂ )r-CONH]s-(CH ₂ )t, wherein r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5;

Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety;

R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ;

Among them, R ₂ and R ₃ are not H at the same time.

As one of the preferred technical solutions, the compound of formula I combined with the blocking agent, wherein,

R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} or -YXQ,

_R3 is selected from -YXQ,

where Y is _CH2 or C=O,

X is O;

or (CH ₂ )n, wherein n is an integer from 1 to 20;

or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30;

or [(CH ₂ )r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5;

Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety;

R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ;

Among them, R ₁ and R ₂ are not H at the same time. As one of the preferred technical solutions, compound Q of formula I in any of the above solutions is ZL, wherein Z is selected from haptens and/or polypeptides that do not contain a biotin moiety.

According to an embodiment of the present invention, when R ₁ is selected from -YXQ, R ₃ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ; and R ₂ and R ₃ are not H at the same time.

According to an embodiment of the present invention, when R ₃ is selected from -YXQ, R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ; and R ₁ and R ₂ are not H at the same time.

According to an embodiment of the present invention, when R ₁ is selected from -YXQ, R ₃ is selected from H or -YXQ; and R ₂ and R ₃ are not H at the same time.

According to an embodiment of the present invention, when R ₃ is selected from -YXQ, R ₁ is selected from H or -YXQ; and R ₁ and R ₂ are not H at the same time.

According to an embodiment of the invention, _R2 is selected from H, methyl, acetyl, methoxyethyl.

According to an embodiment of the present invention, -YX- is selected from: -(CH ₂ )nC(=O)-, [(CH ₂ )rC(=O)NH]s-(CH ₂ )tC(=O)-, [(CH ₂ )pO]k, [(CH ₂ )rC(=O)NH]s-[(CH ₂ )tO]u; n is an integer from 2 to 6, r is an integer from 2 to 6, and t is An integer from 0 to 3, s is an integer from 1 to 3, u is an integer from 2 to 4, p is 2, and k is an integer from 1 to 5.

According to an embodiment of the invention, -YX- is selected from:

According to an embodiment of the present invention, Q is selected from NH ₂ , azido, COOH,

According to an embodiment of the invention, -YXQ is selected from:

According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

As a further preference, the blocking agent is an antibody, preferably a monoclonal antibody. The affinity of the blocker for biotin and its metabolites is at least 50 times higher than the affinity of bound biotin or its derivatives; preferably at least 500 times, 1000 times, 2000 times, 5000 times, 10000 times.

The present invention also provides a compound as shown in formula I for use as an immunogen/intermediate for preparing an immunogen/screening an antibody:

It is characterized in that R ₁ and R ₃ are the same or different, and are independently selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy)-C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl -C(=O)-, C ₁ -C ₃ alkylsulfonyl or -YXQ,

where Y is selected from Absent, _CH2 or C(=O),

X is selected from O;

or (CH ₂ )n and n is an integer from 1 to 20;

or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30;

Or [(CH ₂ )r-CONH]s-(CH ₂ )t, [(CH ₂ )r-CONH]s-[(CH ₂ )tO]u, where r is an integer from 1 to 6 and t is 0 to 5, and s is an integer from 1 to 5, u is an integer from 1 to 5;

Q is H, OH, C(=O)ORq, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety ; Rq is selected from H, succinimide or other active esters;

R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl-C(=O)-, C ₁ -C ₃ alkylsulfonyl;

And when any one of the three substituents R ₁ , R ₂ and R ₃ is selected from H, the remaining two are not H.

As one of the preferred technical solutions, R ₁ is selected from -YXQ,

_R3 is selected from H, _C1 - _C3 alkyl, C1- _C3 haloalkyl, ( _{C1 - C3} alkoxy)-C1 _{- C3} alkyl, _C1 - _C3 acyl, _C1 - _C3 alkylsulfonyl or -YXQ

where Y is _CH2 or C=O,

X is O;

or (CH ₂ )n and n is an integer from 1 to 20;

or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30;

or [(CH2)r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5;

Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety;

R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ;

Among them, R ₂ and R ₃ are not H at the same time.

As one of the preferred technical solutions, R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ,

R ₃ selected from -YXQ

where Y is _CH2 or C=O,

X is O;

or (CH ₂ )n and n is an integer from 1 to 20;

or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30;

or [(CH2)r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5;

Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety;

R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ;

Among them, R ₁ and R ₂ are not H at the same time.

The present invention also provides a method for preparing the above blocking agent, which is characterized in that it includes immunizing animals with the compound of formula I and further screening to obtain antibodies, wherein compound Q of formula I is Z-L, wherein Z is selected from the group consisting of biotin-free parts. of haptens and/or peptides.

Preferably, the above method also includes screening antibodies using biotin and its metabolites as competitors of the compound of formula I, and selecting antibodies that can competitively bind to biotin and its metabolites.

Preferably, the above method further comprises screening the antibodies using bound biotin or its derivatives to select antibodies that do not bind thereto.

Preferably, the antibody includes:

Heavy chain variable region CDR1 as listed in SEQ ID NO:15;

Heavy chain variable region CDR2 as listed in SEQ ID NO:16;

Heavy chain variable region CDR3 as listed in SEQ ID NO:17;

Light chain variable region CDR1 as listed in SEQ ID NO:18;

The light chain variable region CDR2 as set forth in SEQ ID NO:19;

Light chain variable region CDR3 as listed in SEQ ID NO:20;

The above antibody is named Antibody A;

or

The heavy chain variable region CDR1 as set forth in SEQ ID NO:21;

Heavy chain variable region CDR2 as listed in SEQ ID NO:22;

Heavy chain variable region CDR3 as listed in SEQ ID NO:23;

Light chain variable region CDR1 as listed in SEQ ID NO:24;

Light chain variable region CDR2 as listed in SEQ ID NO:25;

Light chain variable region CDR3 as listed in SEQ ID NO:26;

The above antibody is named antibody B;

or

The heavy chain variable region CDR1 as set forth in SEQ ID NO:27;

Heavy chain variable region CDR2 as listed in SEQ ID NO:28;

Heavy chain variable region CDR3 as listed in SEQ ID NO:29;

The light chain variable region CDR1 as set forth in SEQ ID NO:30;

The light chain variable region CDR2 as set forth in SEQ ID NO:31;

Light chain variable region CDR3 as listed in SEQ ID NO:32;

The above antibody is named antibody C;

or

Heavy chain variable region CDR1 as listed in SEQ ID NO:33;

Heavy chain variable region CDR2 as listed in SEQ ID NO:34;

Heavy chain variable region CDR3 as listed in SEQ ID NO:35;

Light chain variable region CDR1 as listed in SEQ ID NO:36;

Light chain variable region CDR2 as listed in SEQ ID NO:37;

The light chain variable region CDR3 as set forth in SEQ ID NO:38;

The above antibody is named antibody D;

or

Heavy chain variable region CDR1 as listed in SEQ ID NO:39;

Heavy chain variable region CDR2 as listed in SEQ ID NO:40;

Heavy chain variable region CDR3 as listed in SEQ ID NO:41;

Light chain variable region CDR1 as listed in SEQ ID NO:42;

Light chain variable region CDR2 as listed in SEQ ID NO:43;

Light chain variable region CDR3 as listed in SEQ ID NO:44;

The above antibody is named antibody E;

or

The heavy chain variable region CDR1 as set forth in SEQ ID NO:45;

Heavy chain variable region CDR2 as listed in SEQ ID NO:46;

Heavy chain variable region CDR3 as listed in SEQ ID NO:47;

The light chain variable region CDR1 as set forth in SEQ ID NO:48;

Light chain variable region CDR2 as listed in SEQ ID NO:49;

The light chain variable region CDR3 as set forth in SEQ ID NO:50;

The above antibody is named antibody F;

or

Heavy chain variable region CDR1 as set forth in SEQ ID NO:51;

Heavy chain variable region CDR2 as listed in SEQ ID NO:52;

The heavy chain variable region CDR3 as set forth in SEQ ID NO:53;

Light chain variable region CDR1 as listed in SEQ ID NO:54;

Light chain variable region CDR2 as listed in SEQ ID NO:55;

Light chain variable region CDR3 as listed in SEQ ID NO:56;

The above antibody was named Antibody G.

beneficial effects

The biotin blocker of the present invention can specifically recognize and combine biotin and its metabolites in samples with high sensitivity, eliminate the interference of biotin and its metabolites on the chemiluminescent biotin-streptavidin system, and ensure Accuracy of test results.

Definitions and explanations of terms

The terms "biotin" or "free biotin" are used interchangeably and refer to the naturally occurring compound, namely, D(+)-biotin. Bound biotin refers to a compound formed by covalently linking the carboxyl group of biotin to other groups. For example, the carboxyl group of biotin forms an amide bond or an ester bond with a compound having an amino or hydroxyl group.

"Biotin metabolites" refer to the products of biotin metabolized in the body, such as bisnorbiotin, tetranorbiotin, biotin sulfoxide, biotin disulfoxide and biotin sulfone.

The term "antibody" or "monoclonal antibody" includes various forms of antibody structures, including, but not limited to, intact antibodies and antibody fragments. The antibody according to the invention is preferably a goat, sheep, mouse, rabbit or rat antibody, a chimeric antibody or a further genetically engineered antibody, as long as the characteristic properties according to the invention are retained. "Antibody fragment" includes a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single chain antibody molecules; scFv, sc(Fv)2; diabodies; and multispecific antibodies formed from antibody fragments.

"Haptens" are small molecules that do not directly induce an immune response, such as the formation of antibodies. Techniques for generating antibodies against haptens by conjugating them to immunogenic carriers, such as antigenic macromolecules, have been established. For the purpose of this disclosure, a hapten is understood to be a low molecular weight molecule, in particular having a molecular weight of 10,000 Da or less, which does not elicit an immune response until and unless conjugated to an immunogenic carrier such as a protein.

The term "linker" refers to a bifunctional or polyfunctional moiety through which two or more moieties are joined "through" the linker. As will be apparent to the skilled person, in such conjugates the functional part of the linker is present as part of the bond and not as unreacted functional part. Preference is given to N-hydroxysuccinimide groups or maleimide groups to bind the linker to amino groups; or groups suitable for binding to second functional groups, for example for reaction with SH-groups, preferably maleimide groups imide group to bind the linker to the SH-group. In a specific embodiment, the heterobifunctional linker is selected from NHS-maleimide linkers based on N-hydroxysuccinimide and maleimide reactive groups; succinimidyl-(PEG)n NHS -PEG-maleimide linker, NHS-haloacetyl linker; and NHS-pyridyldisulfide linker. In a particularly preferred embodiment, the heterobifunctional linker is a succinimidyl-(PEG)NHS-PEG-maleimide linker.

Detailed ways

The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Embodiment 1

Synthesis of Compound 2

Dissolve D-biotin (2.44 g, 10 mmol) in anhydrous methanol (75 mL); then add concentrated sulfuric acid (1 mL); reflux the reaction solution (70°C) for 7 hours under nitrogen protection; cool the reaction solution to room temperature and concentrate it under reduced pressure at 45°C; slurry the residue with methyl tert-butyl ether (75 mL) and filter it, wash the filter cake with a small amount of methyl tert-butyl ether, and dry the solid under reduced pressure at 45°C to obtain about 2 g of white powder. MS Found: [M+H] ⁺ = 259.34

Synthesis of Compound 3

Dissolve compound 2 (1g, 3.87mmol) in pyridine (20mL); add 4,4'-bismethoxytrityl chloride (2g, 6mmol); then add DMAP (50mg, 0.38mmol); fully After mixing and dissolving, transfer the above mixed solution to a 100 mL stainless steel jar lined with PTFE; stir the above reaction solution at 75°C for 18 hours; after cooling to room temperature, pour the reaction solution into 200 mL of deionized water, and use 200 mL of deionized water. Extract twice with ethyl acetate. After combining the organic phases, wash the organic phase once with 200 mL of 1N hydrochloric acid, once with 100 mL of saturated sodium bicarbonate, and once with 100 mL of saturated brine, then dry over anhydrous sodium sulfate, and concentrate under reduced pressure at 45°C. ; The resulting residue was purified by column chromatography to obtain approximately 1.68g of white solid. MS Found：[M+H] ⁺ =561.71

Synthesis of compound 4

Compound 3 (1g, 1.78mmol) was dissolved in THF (20mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice water bath; sodium hydride (60%, 100mg, 2.67mmol) was added to the above solution at one time In the reaction mixture; stir the above reaction mixture at 0°C for 30 minutes; add acetyl chloride (210mg, 2.67mmol) dropwise into the above reaction mixture, and control the internal temperature to be less than 20°C; do not withdraw cooling after the dropwise addition. Bath, stir for 3 hours at 0-20°C; pour the reaction solution into 200 mL of deionized water, extract twice with 200 mL of ethyl acetate, combine the organic phases, wash once with 100 mL of saturated brine, and dry over anhydrous sodium sulfate at 45 °C and concentrated under reduced pressure at 45 °C; the residue was dried under reduced pressure and vacuum at 45 °C to obtain about 1.1 g of yellow solid. MS Found：[M+H] ⁺ ＝603.75

Synthesis of compound 5

Dissolve compound 4 (1g, 1.66mmol) in methanol (20mL); use an ice-water bath to cool to 0°C; add a saturated solution of hydrogen chloride in methanol (20mL); raise the temperature to room temperature naturally without removing the cold bath; stir for 15 hours Afterwards, the reaction solution was concentrated under reduced pressure at 45°C; the resulting residue was purified by column chromatography to obtain approximately 0.34 g of white solid. MS Found：[M+H] ⁺ ＝301.37

Synthesis of compound 6

Compound 5 (0.34g, 1.13mmol) was dissolved in THF (20mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; sodium hydride (60%, 68mg, 1.7mmol) was added in one go. In the above reaction mixture; stir the above reaction mixture at 0°C for 30 minutes; dissolve Fomc-alanine chloride (0.56g, 1.7mmol) in ultra-dry THF (10 mL), and then add it dropwise to the above reaction mixture liquid, control the internal temperature to be less than 20 degrees Celsius; do not remove the cold bath after the dropwise addition, stir at 0-20 degrees Celsius for 3 hours; pour the reaction solution into 200mL deionized water, extract twice with 200mL ethyl acetate, and combine the organic phases , washed once with 100 mL saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; the residue was purified by column chromatography to obtain about 0.5 g of yellow solid. MS Found：[M+H] ⁺ ＝594.75

Synthesis of Compound 7

Compound 6 (0.5 g, 0.84 mmol) was dissolved in methanol (10 mL); the solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; piperidine (0.7 g, 8.4 mmol) was added to the reaction mixture at once; the reaction mixture was stirred at 0-20°C for 3 hours; the reaction solution was concentrated under reduced pressure and vacuum at 45°C; the residue was dried under reduced pressure and vacuum at 45°C; the obtained yellow oily residue was dissolved in methanol (10 mL); lithium hydroxide monohydrate (0.1 g, 2.5 mmol) was added, the reaction mixture was stirred at 60°C for 1.5 hours and then cooled to room temperature; the mixture was concentrated under reduced pressure and vacuum at 45°C; the residue was purified by Pre-HPLC to obtain about 167 mg of white solid. MS Found: [M+H] ⁺ = 358.43

Synthesis of compound 8

Compound 7 (167 mg, 0.46 mmol) was dissolved in DMF (5 mL); disuccinimide suberate (172 mg, 0.46 mmol) was added under nitrogen protection, and the reaction solution was stirred at room temperature under nitrogen protection for 1- 2 hours; the reaction solution was directly purified by Pre-HPLC to obtain about 98 mg White solid. MS Found：[M+H] ⁺ ＝611.68

Embodiment 2

Synthesis of Compound 9

Compound 3 (1g, 1.78mmol) was dissolved in THF (20mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice water bath; sodium hydride (60%, 100mg, 2.67mmol) was added to the above solution at one time In the reaction mixture; stir the above reaction mixture at 0°C for 30 minutes; add methyl iodide (380mg, 2.67mmol) dropwise into the above reaction mixture, and control the internal temperature to be less than 20°C; do not withdraw cooling after the dropwise addition. Bath, stir for 3 hours at 0-20°C; pour the reaction solution into 200 mL of deionized water, extract twice with 200 mL of ethyl acetate, combine the organic phases, wash once with 100 mL of saturated brine, and dry over anhydrous sodium sulfate at 45 °C and concentrated under reduced pressure at 45 °C; the residue was dried under reduced pressure and vacuum at 45 °C to obtain about 1.1 g of yellow solid. MS Found：[M+H] ⁺ =575.75

Synthesis of compound 10

Dissolve compound 9 (1g, 1.66mmol) in methanol (20mL); use an ice-water bath to cool to 0°C; add a saturated solution of hydrogen chloride in methanol (20mL); raise the temperature to room temperature naturally without removing the cold bath; stir for 15 hours Afterwards, the reaction solution was concentrated under reduced pressure at 45°C; the resulting residue was purified by column chromatography to obtain approximately 0.39 g of white solid. MS Found：[M+H] ⁺ =273.37

Synthesis of compound 11

Compound 10 (0.39g, 1.42mmol) was dissolved in THF (20mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; sodium hydride (60%, 86mg, 2.14mmol) was added in one go. In the above reaction mixture; stir the above reaction mixture at 0°C for 30 minutes; dissolve Fomc-alanine acyl chloride (0.56g, 1.7mmol) in ultra-dry THF (10 mL), and then add it dropwise to the above reaction mixture liquid, control the internal temperature to be less than 20 degrees Celsius; do not remove the cold bath after the dropwise addition, stir at 0-20 degrees Celsius for 3 hours; pour the reaction solution into 200mL deionized water, extract twice with 200mL ethyl acetate, and combine the organic phases , washed once with 100 mL saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; The residue was purified by column chromatography to obtain about 0.5 g of yellow solid. MS Found：[M+H] ⁺ ＝566.69

Synthesis of compound 12

Compound 11 (0.5g, 0.88mmol) was dissolved in methanol (10mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; piperidine (0.74g, 8.8mmol) was added to the above reaction in one go in the mixed solution; stir the above reaction mixture at 0-20°C for 3 hours; concentrate the reaction solution under reduced pressure and vacuum at 45°C; dry the residue under reduced pressure and vacuum at 45°C; dissolve the resulting yellow oily residue in methanol (10mL); add lithium hydroxide monohydrate (0.1g, 2.5mmol), stir the reaction mixture at 60°C for 1.5 hours and then cool to room temperature; concentrate the above mixture under reduced pressure at 45°C in vacuo; the residue is purified with Pre - HPLC purification, obtaining approximately 136 mg of white solid. MS Found：[M+H] ⁺ =330.43

Synthesis of Compound 13

Compound 12 (136 mg, 0.41 mmol) was dissolved in DMF (5 mL); disuccinimidyl suberate (151 mg, 0.41 mmol) was added under nitrogen protection, and the reaction solution was stirred at room temperature for 1- 2 hours; the reaction solution was directly purified by Pre-HPLC to obtain about 88 mg of white solid. MS Found：[M+H] ⁺ =583.68

Embodiment 3

Synthesis of compound 14

Compound 3 (1g, 1.78mmol) was dissolved in THF (20mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice water bath; sodium hydride (60%, 100mg, 2.67mmol) was added to the above solution at one time into the reaction mixture; stir the above reaction mixture at 0°C for 30 minutes; add potassium iodide (0.44g, 2.67mmol) into the above reaction system at one time, and then add 2-bromoethyl methyl ether (371mg, 2.67mmol) mmol) was added dropwise to the above reaction mixture, and the internal temperature was controlled to be less than 20 degrees Celsius; after the dropwise addition, the cold bath was not withdrawn, and stirred at 0-20 degrees Celsius for 3 hours; the reaction solution was poured into 200mL deionized water, and 200mL ethyl acetate was used. The ester was extracted twice, the organic phases were combined, washed once with 100 mL saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; the residue was dried under reduced pressure at 45°C to obtain about 1.2g of yellow solid. MS Found：[M+H] ⁺ ＝619.75

Synthesis of compound 15

Dissolve compound 14 (1g, 1.61mmol) in methanol (20mL); use an ice-water bath to cool to 0°C; add a saturated solution of hydrogen chloride in methanol (20mL); without removing the cold bath, warm to room temperature naturally; stir for 15 hours. , the reaction solution was concentrated under reduced pressure at 45°C; the resulting residue was purified by column chromatography to obtain about 0.36g of white solid. MS Found：[M+H] ⁺ =317.37

Synthesis of compound 16

Compound 15 (0.36g, 1.13mmol) was dissolved in THF (20mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; sodium hydride (60%, 68mg, 1.7mmol) was added in one go. In the above reaction mixture; stir the above reaction mixture at 0°C for 30 minutes; dissolve Fomc-alanine chloride (0.56g, 1.7mmol) in ultra-dry THF (10 mL), and then add it dropwise to the above reaction mixture liquid, control the internal temperature to be less than 20 degrees Celsius; do not remove the cold bath after the dropwise addition, stir at 0-20 degrees Celsius for 3 hours; pour the reaction solution into 200mL deionized water, extract twice with 200mL ethyl acetate, and combine the organic phases , washed once with 100 mL saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 45°C; the residue was purified by column chromatography to obtain about 0.5 g of yellow solid. MS Found：[M+H] ⁺ ＝610.69

Synthesis of compound 17

Compound 16 (0.5g, 0.82mmol) was dissolved in methanol (10mL); the above solution was protected with nitrogen and then cooled to 0°C in an ice-water bath; piperidine (0.74g, 8.8mmol) was added to the above reaction in one go in the mixed solution; stir the above reaction mixture at 0-20°C for 3 hours; concentrate the reaction solution under reduced pressure and vacuum at 45°C; dry the residue under reduced pressure and vacuum at 45°C; dissolve the resulting yellow oily residue in methanol (10mL); add lithium hydroxide monohydrate (0.1g, 2.5mmol), stir the reaction mixture at 60°C for 1.5 hours and then cool to room temperature; concentrate the above mixture under reduced pressure at 45°C in vacuo; the residue is purified with Pre - HPLC purification, obtaining approximately 144 mg of white solid. MS Found：[M+H] ⁺ ＝374.43

Synthesis of compound 18

Compound 17 (144 mg, 0.38 mmol) was dissolved in DMF (5 mL); disuccinimidyl suberate (141 mg, 0.38 mmol) was added under nitrogen protection, and the reaction solution was stirred at room temperature under nitrogen protection for 1- 2 hours; the reaction solution was directly purified by Pre-HPLC to obtain about 69 mg White solid. MS Found：[M+H] ⁺ ＝627.68

Embodiment 4

Synthesis of compound 20

Compound 19 (19.4g, 0.1mol) was dissolved in dichloromethane (200mL), triethylamine (3g, 0.3mol) was added, and the reaction mixture was cooled to 0°C in an ice-water bath; p-toluenesulfonyl chloride (9.5 g, 0.5 mmol) was dissolved in dichloromethane (50 mL), and slowly added dropwise to the above mixture; without removing the cold bath after the dropwise addition, the above reaction mixture was stirred at 0-20°C for 3 hours; the reaction mixture was Pour into 250mL deionized water, separate the liquids, wash the organic phase once with 100mL 2N hydrochloric acid, once with 100mL saturated sodium bicarbonate, and once with saturated brine, then dry with anhydrous sodium sulfate and concentrate under reduced pressure at 45°C; The residue was purified by column chromatography to obtain 12 g of light yellow oil.

Synthesis of Compound 21

Compound 21 (12g, 34.5mmol) was dissolved in DMF (50mL), sodium azide (2.5g, 38.4mmol) was carefully added, the reaction mixture was heated to 80°C, and stirred at 80°C for 15 hours; the reaction was After the mixture was cooled to room temperature, 200 mL of ethyl acetate was added, and the insoluble white solid was carefully filtered off. The filtrate was concentrated to dryness under reduced pressure at 45°C. The residue was purified by column chromatography to obtain 6.87 g of light yellow oil.

Synthesis of compound 22

Compound 5 (0.34g, 1.13mmol) was dissolved in methanol (10mL), and sodium periodate (0.36g, 1.7mmol) was added; the above mixture was stirred at room temperature for 15 hours and then concentrated under reduced pressure at 45°C; the residue was Purification by column chromatography yielded 215 mg of light yellow solid. MS Found：[M+H] ⁺ ＝317.68

Synthesis of compound 23

Compound 22 (215 mg, 0.68 mmol) was dissolved in dry chloroform (5 mL), and the mixture was cooled to -60°C in a dry ice acetone bath under nitrogen protection, and trifluoroacetic anhydride (300 uL, 2.1 mmol) was added; the reaction mixture was stirred at -60°C for 30 minutes, the cold bath was removed, the temperature was naturally raised to room temperature, and then stirred at room temperature for 30 minutes; the reaction mixture was concentrated under reduced pressure and vacuum at 30°C, and then dried under high vacuum at room temperature for 2 hours; the residual light red oil was dissolved in ultra-dry tetrahydrofuran (3 mL), and compound 21 (0.8 g, 3.65 mmol) was added; the reaction mixture was stirred at room temperature for 48 hours under nitrogen protection; filtered, the insoluble solid was filtered out, and the filtrate was directly purified by Pre-HPLC to obtain 36 mg of white solid. MS Found: [M+H] ⁺ = 518.68

Synthesis of compound 24

Compound 23 (36 mg, 0.07 mmol) was dissolved in methanol (10 mL); lithium hydroxide monohydrate (5 mg, 0.12 mmol) was added, the reaction mixture was stirred at 60°C for 30 minutes and then cooled to room temperature; 2 drops of acetic acid were added to acidify The reaction mixture was then concentrated under reduced pressure at 45°C; the white solid obtained was used directly in the next step. MS Found：[M+H] ⁺ ＝504.68

Synthesis of compound 25

Dissolve the crude compound 23 obtained in the previous step in methanol (10 mL); add palladium on carbon (3 mg); stir the above mixture at room temperature for 1.5 hours under a hydrogen atmosphere pressure of 1 atm; filter, and concentrate the filtrate under reduced pressure at 45°C; The white solid obtained was used directly in the next step. MS Found：[M+H] ⁺ ＝478.68

Synthesis of compound 26

Dissolve the crude compound 23 obtained in the previous step in DMF (5 mL), add disuccinimide suberate (26 mg, 0.07 mmol) under nitrogen protection, and stir the reaction solution at room temperature for 1-2 hours under nitrogen protection. ; The reaction solution was directly purified by Pre-HPLC to obtain about 11 mg of white solid. MS Found：[M+H] ⁺ =731.68

Embodiment 5

Synthesis of compound 27

Compound 10 (0.39g, 1.42mmol) was dissolved in methanol (10mL), and sodium periodate (0.46g, 2.13mmol) was added; the above mixture was stirred at room temperature for 15 hours and then concentrated under reduced pressure at 45°C; the residue was Purification by column chromatography yielded 215 mg of light yellow solid. MS Found：[M+H] ⁺ =289.68

Synthesis of compound 28

Compound 27 (215 mg, 0.74 mmol) was dissolved in dry chloroform (5 mL). The above mixture was cooled to -60°C using a dry ice acetone bath under nitrogen protection, and trifluoroacetic anhydride (300 uL, 2.1 mmol) was added; the reaction mixture was After stirring at -60°C for 30 minutes, remove the cold bath, naturally warm to room temperature, and then stir at room temperature for 30 minutes; the reaction mixture is vacuum concentrated under reduced pressure at 30°C, and then dried under high vacuum at room temperature for 2 hours; the remaining light red color The oil was dissolved in ultra-dry tetrahydrofuran (3 mL), and compound 21 (0.8 g, 3.65 mmol) was added; the reaction mixture was stirred at room temperature for 48 hours under nitrogen protection; filtered to remove the insoluble solid, and the filtrate was directly purified by Pre-HPLC. , 34 mg of white solid was obtained. MS Found：[M+H] ⁺ ＝490.68

Synthesis of compound 29

Compound 28 (34 mg, 0.07 mmol) was dissolved in methanol (10 mL); lithium hydroxide monohydrate (5 mg, 0.12 mmol) was added, the reaction mixture was stirred at 60°C for 30 minutes and then cooled to room temperature; 2 drops of acetic acid were added to acidify The reaction mixture was then concentrated under reduced pressure at 45°C; the white solid obtained was used directly in the next step. MS Found：[M+H] ⁺ ＝476.68

Synthesis of compound 30

Dissolve the crude compound 29 obtained in the previous step in methanol (10 mL); add palladium on carbon (3 mg); stir the above mixture at room temperature for 1.5 hours under a hydrogen atmosphere pressure of 1 atm; filter, and concentrate the filtrate under reduced pressure at 45°C; The white solid obtained was used directly in the next step. MS Found：[M+H] ⁺ ＝450.68

Synthesis of Compound 31

Dissolve the crude compound 30 obtained in the previous step in DMF (5 mL), add disuccinimide suberate (26 mg, 0.07 mmol) under nitrogen protection, and stir the reaction solution at room temperature for 1-2 hours under nitrogen protection. ; The reaction solution was directly purified by Pre-HPLC to obtain about 11 mg of white solid. MS Found：[M+H] ⁺ ＝703.68

Embodiment 6

Synthesis of compound 32

Compound 10 (0.36g, 1.13mmol) was dissolved in methanol (10mL), and sodium periodate (0.46g, 2.13mmol) was added; the above mixture was stirred at room temperature for 15 hours and then concentrated under reduced pressure at 45°C; the residue was Purification by column chromatography yielded 207 mg of light yellow solid. MS Found：[M+H] ⁺ ＝333.68

Synthesis of compound 33

Compound 32 (207 mg, 0.62 mmol) was dissolved in dry chloroform (5 mL), and the mixture was cooled to -60°C in a dry ice acetone bath under nitrogen protection, and trifluoroacetic anhydride (300 uL, 2.1 mmol) was added; the reaction mixture was stirred at -60°C for 30 minutes, the cold bath was removed, the temperature was naturally raised to room temperature, and then stirred at room temperature for 30 minutes; the reaction mixture was concentrated under reduced pressure and vacuum at 30°C, and then dried under high vacuum at room temperature for 2 hours; the residual light red oil was dissolved in ultra-dry tetrahydrofuran (3 mL), and compound 21 (0.8 g, 3.65 mmol) was added; the reaction mixture was stirred at room temperature for 48 hours under nitrogen protection; filtered, the insoluble solid was filtered out, and the filtrate was directly purified by Pre-HPLC to obtain 42 mg of white solid. MS Found: [M+H] ⁺ = 534.68

Synthesis of compound 34

Compound 33 (42 mg, 0.08 mmol) was dissolved in methanol (10 mL); lithium hydroxide monohydrate (5 mg, 0.12 mmol) was added, the reaction mixture was stirred at 60°C for 30 minutes and then cooled to room temperature; 2 drops of acetic acid were added to acidify The reaction mixture was then concentrated under reduced pressure at 45°C; the white solid obtained was used directly in the next step. MS Found：[M+H] ⁺ =520.68

Synthesis of compound 35

The crude compound 34 obtained in the previous step was dissolved in methanol (10 mL); palladium carbon (3 mg) was added; the mixture was stirred at room temperature for 1.5 hours under 1 atm hydrogen atmosphere pressure; filtered, and the filtrate was concentrated under reduced pressure at 45°C; the obtained white solid was directly used in the next step. MS Found: [M+H] ⁺ = 494.68

Synthesis of compound 36

The crude compound 35 obtained in the previous step was dissolved in DMF (5 mL), disuccinimidyl suberate (30 mg, 0.08 mmol) was added under nitrogen protection, and the reaction solution was stirred at room temperature for 1-2 hours under nitrogen protection; the reaction solution was directly purified by Pre-HPLC to obtain about 14 mg of white solid. MS Found: [M+H] ⁺ = 747.68

Embodiment 7

Synthesis of compound 37

Dissolve D-biotin (2.44g, 10mmol) in ultra-dry tetrahydrofuran (20mL), add imidazole (2g, 30mmol), protect the above reaction mixture with nitrogen and cool it to 0°C in an ice-water bath; add triphenylsilyl chloride (3.83g, 15mmol) was dissolved in ultra-dry tetrahydrofuran (10mL) and added dropwise to the above reaction mixture; keep stirring at 0°C for 30 minutes; add triethylamine (3g, 30mmol) to the above reaction mixture in one go. Then dissolve Fomc-alanine acyl chloride (3.6g, 11mmol) in ultra-dry THF (10mL) and add it dropwise to the above reaction mixture, controlling the internal temperature to be less than 20 degrees Celsius; do not remove the cold bath after the dropwise addition. Stir at 0-20°C for 3 hours; pour the reaction solution into 200 mL of 1N hydrochloric acid in water, extract twice with 200 mL of ethyl acetate, combine the organic phases, wash once with 100 mL of saturated brine, and dry over anhydrous sodium sulfate at 45°C Concentrate under reduced pressure; the residue is purified by column chromatography to obtain about 1.5 g of white solid. MS Found：[M+H] ⁺ ＝538.69

Synthesis of compound 38

Dissolve compound 38 (1g, 1.8mmol) in anhydrous methanol (75mL); then add concentrated sulfuric acid (1mL); reflux the above reaction solution (70°C) under nitrogen protection for 7 hours; cool the reaction solution to room temperature and then Concentrate under reduced pressure at 45°C; beat the obtained residue with methyl tert-butyl ether (75 mL) and filter, wash the filter cake with a small amount of methyl tert-butyl ether, and dry the obtained solid under reduced pressure and vacuum at 45°C to obtain about 1g of white color powder. MS Found：[M+H] ⁺ =552.64

Synthesis of compound 39

Compound 38 (1g, 1.8mmol) was dissolved in methanol (10mL), and sodium periodate (0.46g, 2.13mmol) was added; the above mixture was stirred at room temperature for 15 hours and then concentrated under reduced pressure at 45°C; the residue was columned After chromatography purification, 515 mg of light yellow solid was obtained. MS Found：[M+H] ⁺ =568.68

Synthesis of compound 40

Compound 39 (350 mg, 0.62 mmol) was dissolved in dry chloroform (5 mL). The above mixture was cooled to -60°C using a dry ice acetone bath under nitrogen protection, and trifluoroacetic anhydride (300 uL, 2.1 mmol) was added; the reaction mixture was After stirring at -60°C for 30 minutes, remove the cold bath, naturally warm to room temperature, and then stir at room temperature for 30 minutes; the reaction mixture is vacuum concentrated under reduced pressure at 30°C, and then dried under high vacuum at room temperature for 2 hours; the remaining light red color The oil was dissolved in ultra-dry tetrahydrofuran (3 mL), and compound 21 (0.8 g, 3.65 mmol) was added; the reaction mixture was stirred at room temperature for 48 hours under nitrogen protection; filtered to remove the insoluble solid, and the filtrate was directly purified by Pre-HPLC. , 86 mg of white solid was obtained. MS Found：[M+H] ⁺ ＝769.68

Synthesis of Compound 41

Compound 10 (86 mg, 0.11 mmol) was dissolved in methanol (10 mL); palladium on carbon (3 mg) was added; the above mixture was stirred at room temperature for 1.5 hours under a hydrogen atmosphere pressure of 1 atm; filtered, and the filtrate was concentrated under reduced pressure at 45°C. ; Use the obtained white solid directly for the next step. MS Found：[M+H] ⁺ =743.68

Synthesis of compound 42

Dissolve the crude compound 41 from the previous step in methanol (10 mL); protect the above solution with nitrogen and cool it to 0°C in an ice-water bath; add piperidine (28 mg, 0.33 mmol) to the above reaction mixture at one time; Stir the above reaction mixture at 0-20°C for 3 hours; concentrate the reaction solution under reduced pressure at 45°C; dry the residue under reduced pressure at 45°C; dissolve the resulting yellow oily residue in methanol (10 mL) Lithium hydroxide monohydrate (15 mg, 0.33 mmol) was added, and the reaction mixture was stirred at 60°C for 1.5 hours and then cooled to room temperature; the above mixture was concentrated under reduced pressure at 45°C in vacuo; the residue was purified by Pre-HPLC to obtain About 28mg white solid. MS Found：[M+H] ⁺ ＝507.43

Synthesis of compound 43

Compound 42 (28 mg, 0.055 mmol) was dissolved in DMF (5 mL), disuccinimidyl suberate (60 mg, 0.16 mmol) was added under nitrogen protection, and the reaction solution was stirred at room temperature under nitrogen protection for 1- 2 hours; the reaction solution was directly purified by Pre-HPLC to obtain about 20 mg of white solid. MS Found：[M+H] ⁺ ＝1014.68

Example 8 Preparation of Antigen Conjugates

Dissolve 10 mg of the compounds synthesized in Examples 1-7 in 1500 μl DMSO, and add them to 100 mg KLH (keyhole hemocyanin) solution. The pH was adjusted to pH=8.3 and the solution was stirred overnight. Seven groups of antigen conjugates were purified and named antigen conjugates 1-7, and amino groups were analyzed. Antigen:KLH was about 300-500:1.

Example 9 Antibody preparation and screening

1. Rabbit immunity and antibody detection

Freund's complete adjuvant and the antigen conjugate Biotin-KLH protein of Example 8 with a concentration of 2 mg/ml were mixed and emulsified in equal volumes. Four healthy New Zealand white rabbits about 2 months old and weighing 1.5 kg were selected. Rabbits were initially immunized using five-point injection, and the immunization dose was 800ug/animal. Rabbits were immunized weekly during the first month of immunization and monthly starting from the second month. Freund's adjuvant was used for the primary immunization, and incomplete Freund's adjuvant was used for the remaining immunizations. The dosage of immunogen for the booster immunization was half of that for the primary immunization. After 36 days, blood was collected from rabbit ear veins, and the supernatant was collected by centrifugation to detect serum titers using ELISA. Monitor the rabbit serum titer. When the rabbit serum titer is qualified, lymphocytes will be separated and used for cell fusion.

2. Hybridoma cell fusion

Collect the ear arterial blood of the rabbits after immunization, and measure the immune titer by ELISA; sacrifice the rabbits whose titer reaches the target, remove the spleen, grind it, and lyse it to prepare B cells. Rabbit B cells and myeloma cells SP2/0 were mixed at a ratio of 1:1, and then electrofusion was performed to obtain rabbit-mouse hybridoma cells. Then hybridoma cells were added to 1.2% methylcellulose semi-solid medium, HAT, anti-mycoplasma drugs, and feeder cells were added, mixed thoroughly, and plated. Place the cells in an incubator for 3-4 days and then add DMEM complete medium. After 13 days of continuous culture, take the cell supernatant for ELISA testing (preliminary screening and review).

3. Antibody screening

This embodiment takes the detection of serum amyloid A (SAA) as an example. In practical applications, detection markers can be selected according to needs and are not limited to SAA.

In the antibody screening stage, in the system that uses the biotin-avidin system to detect SAA, free biotin is added to interfere with the system signal, and hybridoma clones that can restore the system signal value are screened.

Specific experimental plan: 1ug/ml SAA antigen was coated at 4℃ for 8 hours; after washing, 100ul biotin-labeled SAA antibody (50ng/ml, prepared with 1% BSA+0.1% PBST to achieve blocking effect) was used for incubation for 30min;

Control group 1: add 100ul blank culture medium;

Control group 2: Add 50ul 50ng/ml biotin and 50ul blank culture medium;

Experimental group: Add 50ul of 50ng/ml biotin and 50ul of cell line supernatant;

Add 50ul 1% BSA+0.1% PBST to each of the above three groups; use avidin-HRP (1:5000 times dilution) 100ul/well, incubate at 37°C Incubate for 30 minutes; wash the plate, use chromogenic solution to develop for 3 minutes, stop, and use a microplate reader to read at OD450.

There was no biotin interference in control group 1, so the signal value remained at a high level (greater than 1.5). In control group 2, after using 50ng/ml biotin to interfere with the biotin-avidin system, the signal value was significantly reduced (less than 0.5). In the experimental group Since the addition of cell line supernatant antibodies can bind biotin and exert varying degrees of anti-interference effects, significantly increasing the signal value, the cell lines with the best anti-interference ability (signal value above 1.2) are selected for subsequent experiments.

In this example, antigen conjugates 1-7 are used for immunization. Each antigen conjugate is screened according to the above experimental protocol to select a cell line with the best anti-interference ability.

In the above experiment, the antigen was coated on an enzyme-labeled plate, which was made of polystyrene. The binding with the antigen relied on adsorption or hydrophobic interaction, and would not affect the active groups of the antigen itself.

4. Antibody preparation

Hybridoma cell sequencing. Take the hybridoma cells that are positive in the ELISA test, wash them with DPBS and directly lyse them with lysis buffer, and then use reverse transcription reaction to obtain cDNA. The designed rabbit anti-specific light and heavy chain primers, TAQ enzyme and the obtained cDNA samples were used to conduct in vitro amplification experiments to obtain rabbit anti-light and heavy chain PCR products. Then the gel recovery and T vector construction experiments were carried out through the kit, and then the sequencing company sent it for testing.

Antibody fermentation. Take Expi 293 cells in the logarithmic phase of growth, count them with trypan blue, record the viability rate, and inoculate them at a certain proportion into disposable shake flasks and incubate them in an incubator. After 24 hours, take the required number of cells and transfer them to a new fermentation bottle. , mix the calculated ratio of transfection reagent and plasmid solution 1:1, let it stand for a short time, then slowly pour it into a new shake flask and place it in an incubator for incubation. After 18-22 hours, add a certain proportion of feed material, and then The fermentation supernatant was collected after 5-6 days. Seven antibodies were obtained through ProG column affinity chromatography purification, named antibodies A-G. The heavy chain variable regions of antibodies A-G are shown in SEQ ID NO:1-7, and the light chain variable regions of antibodies A-G are shown in SEQ ID Shown in NO:8-14.

5. Verification of antibody performance

①ELISA affinity test

Affinity detection (affinity of antibody and antigen conjugate) experimental steps: Antigen conjugates 1-7 are respectively coated (1ug/ml) and incubated at 4°C for 8 hours; primary antibody (above-mentioned antibodies A-G) incubation: after washing the plate, after purification Antibody (1ug/ml) three times gradient dilution, 100ul/well, incubate at 37°C for 30 minutes; secondary antibody (goat anti-rabbit) incubation: wash the plate, dilute goat anti-rabbit-HRP (1:5000), 100ul/well, 37 Incubate at ℃ for 30 minutes; use chromogenic solution to develop color for 3 minutes, terminate, and use a microplate reader to read at OD450. The results are shown in Table 1.

Table 1 Antibody activity analysis data

The antibody concentration was selected to be 1ug/ml, and the affinities of antibodies A-G binding to antigen conjugates 1-7 were further shown in Table 2.

Table 2 Antibody activity analysis data

In summary, antigen conjugates 1-7 have good affinity with antibodies A-G and are suitable for subsequent experiments.

② ELISA competition method to detect the binding ability of free biotin

According to the above experiments, it can be seen that the affinity effects of antigen conjugates 1-7 and antibodies A-G are not much different and can be selected at will. Therefore, antigen conjugate 1 was selected for further verification in this experiment.

Competition method (antibody and biotin binding ability) experimental steps: Antigen conjugate 1 coating (1ug/ml) and incubation at 4°C for 8 hours; biotin 1ug/mL, 3 times gradient dilution, 60ul/well, antibody diluted to 500ng /mL, add 40ul/well of antibody, and incubate at 37°C for 30 minutes; wash the plate after incubation with secondary antibody (goat anti-rabbit-HRP), dilute it with goat anti-rabbit-HRP (1:5000), add 100ul/well, and incubate at 37°C for 30 minutes; Use chromogenic solution to develop for 3 minutes, stop, and read at OD450 using a microplate reader.

Because the antibody can bind to the whole antigen and free biotin. When there is no free biotin, the antibody can only bind to the whole antigen and be fixed on the plate. When the goat anti-rabbit-HRP binds to the antibody, it will produce a strong fluorescent signal under the action of the substrate. When there is free biotin in the solution, the antibody will bind to the free biotin, and the goat anti-rabbit-HRP will bind to the antibody and develop color under the substrate. Because the free biotin is still in a free state after binding to the antibody, it will be washed away after washing the plate. Therefore, with the addition of free biotin, the luminescent signal will decrease.

The results are shown in Table 3: As the amount of free biotin used increases, the ELISA signal value decreases significantly.

Table 3 Free biotin binding capacity

③ ELISA competitive method to detect the binding ability of bisnorbiotin

Competition method (binding ability of antibody and bisnorbiotin) experimental steps: whole antigen coating (1ug/ml) and incubation at 4°C for 8 hours; bisnorbiotin 1ug/mL, 3 times gradient dilution, 60ul/well, antibody diluted to 500ng/mL, add 40ul/well of antibody, and incubate at 37°C for 30 minutes; wash the plate after incubation with secondary antibody (goat anti-rabbit-HRP), dilute it with goat anti-rabbit-HRP (1:5000), add 100ul/well, and incubate at 37°C for 30 minutes. ;Use chromogenic solution to develop for 3 minutes, stop, and use a microplate reader to read at OD450.

The results are shown in Table 4: As the dosage of bisnorbiotin binding capacity increases, the ELISA signal value decreases significantly.

Table 4 Binorbiotin binding capacity

It can be seen from the above ② and ③ that the antibodies A-G of the present invention can bind free biotin and bisnorbiotin to avoid the interference of free biotin and bisnorbiotin on the biotin-avidin system, and the antibodies A-G can bind free biotin and bisnorbiotin. The binding abilities of biotin and bisnorbiotin are similar.

Example 10 Anti-interference ability test by chemiluminescence method

It can be seen from Example 9 that antibodies A-G have similar binding abilities to free biotin and bisnorbiotin. In practical applications, interference from the two biotins in the sample usually exists at the same time. Therefore, this example uses free biotin. A mixture of biotin and bisnorbiotin was used to test the anti-interference ability of antibodies A-G in practical applications.

Select the anti-Müllerian hormone (AMH) detection kit (chemiluminescence method), add different concentrations of free biotin and bisnorbiotin mixtures to the sample as interference, and add different concentrations of antibodies A-G to the reagent R1 component. , to verify the effectiveness of different concentrations of antibodies in eliminating the interference of free biotin and bisnorbiotin.

Specific experimental plan: use Zhongyuan Huiji AMH detection kit, add different concentrations of free biotin/bisnorbiotin mixed solutions to the sample, add different concentrations of purified antibodies to the R1 component, use Zhongyuan Huiji EXI1800 A fully automated chemiluminescent immunoanalyzer measures sample values.

AMH detection kit: The amount of R1 biotin antibody is 50ul; the amount of R2 enzyme-labeled antibody is 50ul; the amount of avidin magnetic beads is 30ul; the sample volume is 20ul.

The results are shown in Table 5:

Table 5 Anti-interference ability test

The results show that different doses of free biotin/bisnorbiotin mixed solution will cause interference to the AMH detection signal value. The degree of interference increases with the increase in the concentration of the mixed solution. Antibodies A-G can bind to the mixed solution and significantly improve the signal caused by the mixed solution. The value decreases, and as the concentration of purified antibodies A-G increases, its anti-interference ability is also significantly enhanced. Purified antibodies A-G of 0.5 mg/ml can basically resist the interference of the 2400ng/ml mixture, because antibodies A-G have no effect on free biotin and double The binding ability of biotin is equivalent. Therefore, although this example verifies the anti-interference ability of the mixture, it can be shown that 0.5 mg/ml of purified antibodies A-G can resist 2400 ng/ml of free biotin or bisnorbiotin alone. interference.

The above has provided an exemplary description of the embodiments of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the protection scope of the claims of this application.

Claims (11)

Compounds as shown in formula I:
It is characterized in that R ₁ and R ₃ are the same or different, and are independently selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy)-C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl -C(=O)-, C ₁ -C ₃ alkylsulfonyl or -YXQ, where Y is selected from Absent, _CH2 or C(=O), X is selected from O; or (CH ₂ )n and n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 0, 1, 2 or 3, and k is an integer from 1 to 30; or [(CH ₂ )r-CONH]s-(CH ₂ )t, [(CH ₂ )r-CONH]s-[(CH ₂ )tO]u, wherein r is an integer from 1 to 6, t is an integer from 0 to 5, s is an integer from 1 to 5, and u is an integer from 1 to 5; Q is H, OH, C(=O)ORq, _NH2 , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety ; Rq is selected from H, succinimide or other active esters; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl-C(=O)-, C ₁ -C ₃ alkylsulfonyl; And when any one of the three substituents R ₁ , R ₂ and R ₃ is selected from H, the remaining two are not H; Preferably, R ₁ and R ₃ are the same or different, and are independently selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy)-C ₁ -C ₃ Alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ, where Y is _CH2 or C=O, X is O; or (CH ₂ )n and n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30; or [(CH ₂ )r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5; Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ; And when any one of the three substituents R ₁ , R ₂ and R ₃ is selected from H, the remaining two are not H. The compound of claim 1, wherein, R ₁ is selected from -YXQ, _R3 is selected from H, _C1 - _C3 alkyl, C1- _C3 haloalkyl, ( _{C1 - C3} alkoxy)-C1 _{- C3} alkyl, _C1 - _C3 acyl, _C1 - _C3 alkylsulfonyl or -YXQ where Y is _CH2 or C=O, X is O; or (CH ₂ )n and n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30; or [(CH ₂ )r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5; Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ; Among them, R ₂ and R ₃ are not H at the same time. The compound according to claim 1 or 2, characterized in that, R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} or -YXQ, R ₃ selected from -YXQ where Y is _CH2 or C=O, X is O; or (CH ₂ )n and n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30; or [(CH2)r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5; Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ; Among them, R ₁ and R ₂ are not H at the same time; Preferably, when R ₁ is selected from -YXQ, R ₃ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ Alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ; and R ₂ and R ₃ are not H at the same time; Preferably, when R ₃ is selected from -YXQ, R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ Alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ; and R ₁ and R ₂ are not H at the same time; Preferably, when R ₁ is selected from -YXQ, R ₃ is selected from H or -YXQ; and R ₂ and R ₃ are not H at the same time; Preferably, when R ₃ is selected from -YXQ, R ₁ is selected from H or -YXQ; and R ₁ and R ₂ are not H at the same time; Preferably, R ₂ is selected from H, methyl, acetyl, methoxyethyl; Preferably, -YX- is selected from: -(CH ₂ )nC(=O)-, [(CH ₂ )rC(=O)NH]s-(CH ₂ )tC(=O)-, [(CH ₂ )pO]k, [(CH ₂ )rC(=O)NH]s-[(CH ₂ )tO]u; n is an integer from 2 to 6, r is an integer from 2 to 6, t is an integer from 0 to 3 Integers, s is an integer from 1 to 3, u is an integer from 2 to 4, p is 2, and k is an integer from 1 to 5; Preferably, -YX- is selected from: Preferably, Q is selected from NH ₂ , azido, COOH, Preferably, -YXQ is selected from: Preferably, the compound represented by formula (I) is selected from the following structures:


Preferably, the compound represented by formula (I) is selected from the following structures:

The compound of any one of claims 1 to 3, characterized in that the compound Q of formula I is Z-L, wherein Z is selected from haptens and/or polypeptides that do not contain a biotin moiety. A blocker that specifically binds to the compound according to any one of claims 1 to 4, biotin and biotin metabolites, characterized in that the structure of the compound of formula I is as follows:
Wherein, R ₁ and R ₃ are the same or different, and are independently selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy)-C ₁ -C ₃ alkyl Base, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl -C(=O)-, C ₁ -C ₃ alkylsulfonyl or -YXQ, where Y is selected from Absent, _CH2 or C(=O), X is selected from O; or (CH ₂ )n and n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 0, 1, 2 or 3, and k is an integer from 1 to 30; Or [(CH ₂ )r-CONH]s-(CH ₂ )t, [(CH ₂ )r-CONH]s-[(CH ₂ )tO]u, where r is an integer from 1 to 6 and t is 0 to 5, and s is an integer from 1 to 5, u is an integer from 1 to 5; Q is H, OH, C(=O)ORq, _NH2 , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety ; Rq is selected from H, succinimide or other active esters; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkyl-C(=O)-, C ₁ -C ₃ alkylsulfonyl; And when any one of the three substituents R ₁ , R ₂ and R ₃ is selected from H, the remaining two are not H; Preferably, R ₁ and R ₃ are the same or different, and are independently selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy)-C ₁ -C ₃ Alkyl, C ₁ -C ₃ acyl, C ₁ -C ₃ alkylsulfonyl or -YXQ, where Y is _CH2 or C=O, X is O; or (CH ₂ )n and n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30; or [(CH ₂ )r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5; Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ; And when any one of the three substituents R ₁ , R ₂ and R ₃ is selected from H, the remaining two are not H. The blocking agent of claim 5, wherein the blocking agent binds a compound of formula I, wherein, R ₁ is selected from -YXQ, R ₃ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} or -YXQ, where Y is _CH2 or C=O, X is O; or (CH ₂ )n, wherein n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, where p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30; or [(CH ₂ )r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5; Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ; Among them, R ₂ and R ₃ are not H at the same time. The blocking agent of claim 5 or 6, characterized in that the compound of formula I combined with the blocking agent, wherein, R ₁ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} or -YXQ, R ₃ is selected from -YXQ, where Y is _CH2 or C=O, X is O; or (CH ₂ )n, wherein n is an integer from 1 to 20; or [(CH ₂ )pO]k-(CH ₂ )m, wherein p is 2 or 3, m is 2 or 3, and k is an integer from 1 to 30; or [(CH ₂ )r-CONH]s-(CH ₂ )t, where r is an integer from 1 to 5, t is an integer from 0 to 5, and s is an integer from 1 to 5; Q is H, OH, COOH, NH ₂ , azido, maleimide or ZL, where L is a linker and Z is selected from haptens and/or peptides that do not contain a biotin moiety; R ₂ is selected from H, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, (C ₁ -C ₃ alkoxy) -C ₁ -C ₃ alkyl, C ₁ -C ₃ acyl, C ₁ -C _{3alkylsulfonyl} ; Wherein, _R1 and _R2 are not H at the same time. The blocking agent according to any one of claims 5 to 7, characterized in that the blocking agent is an antibody, preferably a monoclonal antibody. The blocking agent according to any one of claims 5 to 8, characterized in that the affinity of the blocking agent for biotin or its metabolites is at least 50 times higher than the affinity of bound biotin or its derivatives. ; Preferably at least 500 times, 1000 times, 2000 times, 5000 times, 10000 times. A method for preparing the blocking agent according to claims 5-9, characterized in that it includes immunizing animals with a compound of formula I and further screening to obtain antibodies, wherein compound Q of formula I is Z-L, wherein Z is selected from the group consisting of biotin-free Part of the hapten and/or peptide. The method of claim 10, further comprising screening antibodies using biotin and its metabolites as competitors for the compound of formula I, and selecting antibodies that can competitively bind to biotin and its metabolites; Preferably, the preparation method further includes screening antibodies using bound biotin or its derivatives to select antibodies that do not bind to it.