CN117229411B

CN117229411B - Monoclonal antibody specifically binding 25-hydroxy vitamin D, application and detection kit thereof

Info

Publication number: CN117229411B
Application number: CN202311497182.3A
Authority: CN
Inventors: 陶言梦; 刘刚; 董万春; 高歌
Original assignee: Nanjing Oukai Biotechnology Co ltd
Current assignee: Nanjing Oukai Biotechnology Co ltd
Priority date: 2023-11-10
Filing date: 2023-11-10
Publication date: 2024-02-02
Anticipated expiration: 2043-11-10
Also published as: CN117229411A

Abstract

The invention relates to a monoclonal antibody or antigen binding fragment thereof for 25-hydroxyvitamin D, related nucleic acid products, cell products, detection reagents or kits thereof, and preparation methods and applications thereof. The monoclonal antibody or the antigen binding fragment thereof can be combined with 25-hydroxy vitamin D with high specificity and high affinity, so that the detection accuracy and sensitivity of 25-hydroxy vitamin D are improved; in addition, the invention also develops a high-performance paired antibody suitable for the double-antibody sandwich method immunodetection, thereby breaking through the limitation of the traditional competition method, realizing the double-antibody sandwich method immunodetection of 25-hydroxy vitamin D and having great clinical application value.

Description

Monoclonal antibody specifically binding 25-hydroxy vitamin D, application and detection kit thereof

Technical Field

The invention relates to the technical field of monoclonal antibody preparation and immunological detection, in particular to a monoclonal antibody specifically combined with 25-hydroxy vitamin D, application thereof and a detection kit.

Background

25-hydroxy vitamin D (also known as 25-OH-VD) is the major form of vitamin D in the body. Vitamin D is steroid derivative, belongs to fat-soluble vitamin, and is cyclopentane polyhydrophenanthrene compound. Vitamin D is mainly synthesized by human skin after ultraviolet irradiation, and is taken in a small amount from food or supplements. Vitamin D not only affects calcium and phosphorus metabolism, but also has wide physiological effects, is an essential substance for maintaining human health and cell growth and development, and is closely related to various diseases. In humans, there are two forms of vitamin D, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol), which are converted to 25-hydroxy vitamin D (25-OH-VD) by hydroxylation in the liver and then to active 1, 25-dihydroxyvitamin D in the kidneys. The level of serum 25-OH-VD can reflect the storage levels of vitamin D in humans and is associated with clinical symptoms of vitamin D deficiency.

The detection of the level of 25-hydroxy vitamin D is beneficial to comprehensively evaluating the vitamin D nutrition status of patients, guiding the clinical early prevention and treatment of osteoporosis, and providing theoretical support for the clinical reasonable and standard vitamin D treatment. The detection of serum 25-hydroxy vitamin D level is of great clinical significance for finding early rickets and timely treatment.

At present, the gold standard for 25-OH-VD detection is mass spectrometry, but the mass spectrometry has higher requirements on instruments and equipment and personnel, the detection flow is more complicated, the cost is higher, and the method is difficult to popularize in hospitals. The immunization method mainly comprises chemiluminescence method, immunofluorescence method and latex turbidimetry method. However, in view of the fact that 25-OH-VD has a too small molecular weight, it is difficult to develop antibodies against different epitopes thereof for immunological detection using the principle of the double antibody sandwich method, so that these immunological methods currently use the principle of the competition method, however, the sensitivity of the competition method immunological detection is low and the clinical compliance is poor.

In view of the above problems in the prior art, development of a monoclonal antibody with high specificity and high affinity for 25-OH-VD is urgently needed to meet the requirements of immunological detection, and in particular, development of a high-performance pairing antibody is particularly important to break through the limitations of the conventional competition method and realize detection of 25-OH-VD by a double-antibody sandwich method.

Disclosure of Invention

Object of the Invention

In view of the problems or needs in the art, it is an object of the present invention to provide a high specificity, high affinity monoclonal antibody or antigen binding fragment thereof directed against 25-hydroxyvitamin D and to develop a high performance paired antibody suitable for a double antibody sandwich immunoassay.

Solution scheme

In order to achieve the above purpose, the invention obtains the monoclonal antibody or antigen binding fragment thereof aiming at 25-hydroxy vitamin D with high specificity and high affinity through a large number of screening, related nucleic acid and cell products, a preparation method and application thereof, and an immunoassay reagent or kit based on the same, thereby providing a foundation for high-specificity and high-sensitivity immunoassay of 25-hydroxy vitamin D.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D, characterized in that the monoclonal antibody or antigen-binding fragment thereof comprises:

(1) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 4, LAS and SEQ ID NO. 6, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(2) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 14, KAS and SEQ ID NO. 16, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(3) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 21, SEQ ID NO. 22 and SEQ ID NO. 23, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 24, WAS and SEQ ID NO. 26, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(4) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NOS: 34, YAS and 36, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(5) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 41, SEQ ID NO. 42 and SEQ ID NO. 43, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 44, RAS and SEQ ID NO. 46, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(6) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53 for HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NOS: 54, YAS and 56, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(7) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 61, SEQ ID NO. 62 and SEQ ID NO. 63, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 64, DAS and SEQ ID NO. 66 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(8) A heavy chain variable region having amino acid sequences shown as SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO: 74, DTS and SEQ ID NO: 76 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(9) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 81, SEQ ID NO. 82 and SEQ ID NO. 83, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 84, AAS and SEQ ID NO. 86, LCDR1, LCDR2 and LCDR3, respectively;

(10) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 91, SEQ ID NO. 92 and SEQ ID NO. 93 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO: 94, LAS and SEQ ID NO: 96 for LCDR1, LCDR2 and LCDR3, respectively.

In a preferred embodiment, the monoclonal antibody or antigen binding fragment thereof comprises:

(1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 8; or alternatively, the first and second heat exchangers may be,

(2) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 17; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,

(3) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 27; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 28; or alternatively, the first and second heat exchangers may be,

(4) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 37; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 38; or alternatively, the first and second heat exchangers may be,

(5) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 47; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 48; or alternatively, the first and second heat exchangers may be,

(6) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 57; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 58; or alternatively, the first and second heat exchangers may be,

(7) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 67; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 68; or alternatively, the first and second heat exchangers may be,

(8) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 77; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 78; or alternatively, the first and second heat exchangers may be,

(9) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 87; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 88; or alternatively, the first and second heat exchangers may be,

(10) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 97; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 98.

In addition, the monoclonal antibody or antigen binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the group consisting of: constant regions of IgG, igA, or IgM antibodies.

In a further preferred embodiment, the monoclonal antibody or antigen binding fragment thereof comprises:

(1) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 9; and a light chain having an amino acid sequence as shown in SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,

(2) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 19; and a light chain having an amino acid sequence as shown in SEQ ID NO. 20; or alternatively, the first and second heat exchangers may be,

(3) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 29; and a light chain having an amino acid sequence as shown in SEQ ID NO. 30; or alternatively, the first and second heat exchangers may be,

(4) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 39; and a light chain having an amino acid sequence as shown in SEQ ID NO. 40; or alternatively, the first and second heat exchangers may be,

(5) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 49; and a light chain having an amino acid sequence as shown in SEQ ID NO. 50; or alternatively, the first and second heat exchangers may be,

(6) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 59; and a light chain having an amino acid sequence as shown in SEQ ID NO. 60; or alternatively, the first and second heat exchangers may be,

(7) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 69; and a light chain having an amino acid sequence as shown in SEQ ID NO. 70; or alternatively, the first and second heat exchangers may be,

(8) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 79; and a light chain having an amino acid sequence as shown in SEQ ID NO. 80; or alternatively, the first and second heat exchangers may be,

(9) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 89; and a light chain having an amino acid sequence as shown in SEQ ID NO. 90; or alternatively, the first and second heat exchangers may be,

(10) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 99; and a light chain having an amino acid sequence as shown in SEQ ID NO. 100.

In the first aspect, the monoclonal antibodies defined in items (1) to (10) are hereinafter referred to as antibodies 1 to 10, respectively.

In some possible embodiments, the antigen binding fragment of the monoclonal antibody is selected from the group consisting of Fab, fab ', F (ab') ₂ Fd, fv, dAb, complementarity determining region fragments, single chain antibodies, human antibodies, chimeric antibodies or bispecific antibodiesA multispecific antibody.

In a second aspect, the present invention provides a polynucleotide encoding a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above. The polynucleotide is not limited to the method of its production and may be obtained using genetic engineering recombinant techniques or chemical synthesis methods.

In a third aspect, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and, optionally, at least one expression regulatory element operably linked to the polynucleotide.

In a fourth aspect, the present invention provides a recombinant vector comprising a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above.

The vector of the present invention may be a cloning vector or an expression vector, and may be, for example, a plasmid, a cosmid, a phage, or the like.

In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

In a fifth aspect, the present invention provides a transformed host cell into which a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect or a recombinant vector as described in the fourth aspect has been transformed;

such host cells include, but are not limited to: prokaryotic cells, such as E.coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The host cell may also be a cell line, such as a 293T cell line.

Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

In a sixth aspect, the present invention provides the use of a monoclonal antibody or antigen binding fragment thereof as described in the first aspect, a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, a recombinant vector as described in the fourth aspect and/or a transformed host cell as described in the fifth aspect, in the preparation of a detection reagent or kit for detecting 25-hydroxyvitamin D.

In a possible embodiment, the sample is a biological sample of the subject, preferably a serum or plasma sample of the subject.

In a seventh aspect, the present invention provides the use of any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect above in the preparation of a detection reagent or kit for detecting 25-hydroxyvitamin D.

Preferably, the two monoclonal antibodies or antigen binding fragments thereof are selected from the group consisting of:

(I) Antibody 1 or antigen-binding fragment thereof, and antibody 2 or antigen-binding fragment thereof;

(II) antibody 3 or antigen-binding fragment thereof, and antibody 4 or antigen-binding fragment thereof;

wherein antibody 1 or an antigen-binding fragment thereof is defined in any of item (1) of the first aspect, antibody 2 or an antigen-binding fragment thereof is defined in any of item (2) of the first aspect, antibody 3 or an antigen-binding fragment thereof is defined in any of item (3) of the first aspect, and antibody 4 or an antigen-binding fragment thereof is defined in any of item (4) of the first aspect.

In an eighth aspect, the present invention provides a detection reagent for detecting 25-hydroxyvitamin D comprising a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, a recombinant vector as described in the fourth aspect above and/or a transformed host cell as described in the fifth aspect above.

Preferably, the detection reagent is a double antibody sandwich immunoassay kit comprising any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect above, more preferably comprising two monoclonal antibodies or antigen binding fragments thereof selected from the group consisting of:

In a possible embodiment, the double-antibody sandwich immunoassay kit comprises an immunochromatographic test strip, which is based on the principle of double-antibody sandwich method, and takes any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect as a capture antibody and a detection antibody respectively;

Preferably, the immunochromatographic test strip uses any one of (1) an antibody 1 or an antigen-binding fragment thereof and (2) an antibody 2 or an antigen-binding fragment thereof as a capture antibody, and the other one as a detection antibody; further preferably, the immunochromatographic test strip uses an antibody 1 or an antigen-binding fragment thereof as a detection antibody and an antibody 2 or an antigen-binding fragment thereof as a capture antibody;

preferably, the immunochromatographic test strip uses any one of (1) an antibody 3 or an antigen-binding fragment thereof and (2) an antibody 4 or an antigen-binding fragment thereof as a capture antibody, and the other one as a detection antibody; further preferably, the immunochromatographic test strip uses an antibody 3 or an antigen-binding fragment thereof as a detection antibody and an antibody 4 or an antigen-binding fragment thereof as a capture antibody;

preferably, the monoclonal antibody or antigen-binding fragment thereof as a detection antibody is labeled with a detectable label; such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials, enzymes (e.g., horseradish peroxidase), and the like.

In a preferred embodiment, the detectable label is a fluorescent microsphere, a latex microsphere or a colloidal gold particle, preferably a colloidal gold particle.

In a ninth aspect, the present invention provides a method of preparing a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the transformed host cell of the fifth aspect described above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.

In a tenth aspect, the present invention provides a method of detecting the presence or level of 25-hydroxyvitamin D in a sample, the method comprising using a monoclonal antibody or antigen binding fragment thereof as described in the first aspect, a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect, an expression vector as described in the fourth aspect, a transformed host cell as described in the fifth aspect and/or a detection reagent or kit as described in the eighth aspect.

In a possible embodiment, the sample includes, but is not limited to, serum, plasma, and the like from a subject.

The method may be used for diagnostic purposes (e.g., the sample is a sample from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

General methods for detecting the presence or level of an antigen of interest in a sample using monoclonal antibodies or antigen binding fragments thereof are well known to those skilled in the art. In certain preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method, and the like.

Advantageous effects

The monoclonal antibody aiming at the 25-hydroxy vitamin D can be combined with the 25-hydroxy vitamin D with high affinity and high specificity (the combination activity EC50 is less than 15ng/ml, and KD reaches 10 ^-11 M), high-accuracy and high-sensitivity detection of 25-hydroxy vitamin D is realized; in addition, the invention also develops a high-performance pairing antibody suitable for the immunological detection of the double-antibody sandwich method, thereby breaking through the limitation of the traditional competition method, realizing the immunological detection of the double-antibody sandwich method of 25-hydroxy vitamin D, and the detection method has higher specificity and sensitivity, and the clinical coincidence rate is more than 0.95 and is obviously higher than that of the traditional Roche competition method.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a gel diagram of SDS-PAGE detection of 10 anti-25-OH-VD monoclonal antibodies expressed and purified in example 3 of the present invention; wherein M represents the electrophoresis band of molecular weight Marker, lane 1 shows the electrophoresis band of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoresis band of each monoclonal antibody under non-reducing conditions.

FIG. 2 is a graph showing the results of detection of the binding activity of 10 anti-25-OH-VD monoclonal antibodies expressed and purified in example 3 and 25-OH-VD conjugated protein according to the present invention, as detected in example 4.

FIG. 3 is a schematic diagram of a colloidal gold immunochromatographic test strip prepared in example 5 of the present invention; wherein 1 is a PVC bottom plate; 2 is a sample pad; 3 is a nitrocellulose membrane; 4 is gold thread; 5 is a detection line; 6 is a quality control line; and 7 is a water absorption pad.

FIG. 4 is a graph showing the correlation between clinical samples of two colloidal gold immunochromatographic test strips vs. Rogowski competition assay reagents based on two paired antibodies of the present invention, as detected in example 6; fig. 4A is a graph showing the result of the colloidal gold immunochromatographic strip 1 based on the VD mAb1+vd mAb2 paired antibody, fig. 4B is a graph showing the result of the colloidal gold immunochromatographic strip 2 based on the VD mAb3+vd mAb4 paired antibody, and fig. 4C is a graph showing the result of the rogue competition assay reagent.

Detailed Description

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order that the invention may be more readily understood, certain technical and scientific terms are defined as follows. Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ± 5%, ±2%, ±1% and ± 0.1%, as these variations are suitable for carrying out the disclosed methods.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

As used herein, the term "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be construed as inclusive, i.e., including at least one of the list of elements or amounts, but also including more than one, and optionally, additional unlisted items. To the extent that only one term is explicitly recited, such as "only one" or "exactly one" or "consisting of" is used in the claims, it will refer to only one number listed or an element of a list.

The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

"antigen binding fragment" refers to antigen binding fragments of antibodies and antibody analogs, which generally comprise at least a portion of the antigen binding or variable regions of the parent antibody, e.g., one or more CDRs. Fragments of the antibodies retain at least some of the binding specificity of the parent antibody. Antigen binding fragments include peptides selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, diabodies, CDRs comprising peptides, and the like.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains.

The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

"Fab ' fragments" contain a portion of one light chain and one heavy chain comprising a portion of the VH domain, CH1 domain and constant region between the CH1 and CH2 domains, with an inter-chain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.

"F (ab') 2 fragments" contain two light chains and portions of two heavy chains comprising portions of the VH domain, CH1 domain, and constant region between CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Single chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. In general, scFv polypeptides comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

A "diabody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is so short that it is not possible to pair between two domains of the same strand, the domains pair with complementary domains of the other strand and form two antigen binding sites.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

The term "high affinity" for IgG antibodies refers to a KD of 1.0X10 for antigen ^-9 M or lower. For other antibody subtypes, "high affinity" binding may vary.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

Example 1: preparation of monoclonal antibodies against 25-OH-VD

(1) Animal immunization:

taking coupling protein 25-OHDD-BSA (purchased from Nanjing Ming Biotechnology Co., ltd.) as antigen, fully emulsifying with equal amount of Freund's complete adjuvant, subcutaneously injecting to immunize rabbits (variety: new Zealand white rabbits, female, 6-8 weeks old), performing booster immunization at two week intervals for 3 times, taking blood from rabbit vein after the last booster immunization, detecting serum titer by ELISA method, and preparing Peripheral Blood Mononuclear Cell (PBMC) suspension when the serum titer reaches the standard of > 128K;

(2) PBMC preparation:

adding lymphocyte separation liquid Ficoll into a centrifuge tube, taking anticoagulated peripheral blood (whole blood) and sterile PBS, fully mixing uniformly according to a ratio of 1:1-1:5, slowly superposing on a layered liquid level along a tube wall by using a pipette, horizontally centrifuging for 400g multiplied by 30 minutes, removing part of upper liquid after centrifuging, and inserting the rest about 1mL into a cloud layer by using a pipette, and sucking mononuclear cells. Placing into another centrifuge tube, adding more than 5 times of PBS, centrifuging for 300g×10 min, and washing the cells twice. After the last centrifugation, the supernatant was discarded, red blood cell lysate was added, incubated at room temperature for 2 minutes, red blood cells were lysed, 10ml pbs was added, and the cells were washed twice by centrifugation for 300g×10 minutes. The supernatant was discarded, RPMI1640 containing 10% FBS was added, the cells were resuspended, and counted;

(3) Obtaining single B cells:

cell screening was performed using a single cell chip screening platform by using prepared PBMCs, through antigen markers, to obtain B cells with antigen specificity.

Through the steps, 15 monoclonal antibodies aiming at 25-OH-VD are obtained in total, and are identified as IgG antibodies, and are respectively called VD mAb 1-VD mAb15. In the following examples, the light and heavy chain gene sequences of 10 monoclonal antibodies were detected, and expression, purification and binding activity detection were performed.

Example 2: acquisition of light and heavy chain Gene sequences of monoclonal antibodies against 25-OH-VD

Preparation of cDNA:

respectively extracting RNA from 15B cells obtained in the separation in the example 1, carrying out reverse transcription by taking the RNA as a template to obtain cDNA, and amplifying genes of light chains and heavy chains of the antibody by using a PCR (polymerase chain reaction) mode;

acquisition of light and heavy chain genes:

amplifying by utilizing a rabbit-source specific primer, cloning the amplified DNA sequences into vectors, then carrying out conversion coating, picking cloning and extracting plasmids for sequencing to obtain light chain gene sequences and heavy chain gene sequences of 15 antibodies; deducing and obtaining the amino acid sequences of the light chain and the heavy chain of each antibody according to the detected light chain gene sequences and the heavy chain gene sequences of each antibody; specifically, the heavy chain amino acid sequences of the antibodies VD mAb 1-VD mAb15 are respectively shown as SEQ ID NO 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 35 and 125, and the light chain amino acid sequences are respectively shown as SEQ ID NO 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 45 and 135;

Analysis of light and heavy chain variable region gene sequences:

and analyzing the VH and VL according to the sequencing result to finally obtain the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region.

The amino acid sequences of the heavy chain variable region and the light chain variable region of the 15 monoclonal antibodies are shown below, wherein the CDR regions thereof are underlined.

VD mAb1：

VH （SEQ ID NO:7）

QSVEESGGRLVTPGGTLTLTCTASGFTISSNHMCWVRQAPGKGLEYIGFINTGGRAYYASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGAYSSDLWGQGTLVTVSS；

VL （SEQ ID NO:8）

DIVMTQTPASVSEPVGGTVTIKCQASQSISNNLAWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQNNYGSSYGFGGGTGVVVK；

VD mAb2 ：

VH （SEQ ID NO:17）

QSVEESGGRLVTPGGSLTLTCTVSGFSLSSYAMSWVHQAPGKGLEWIGIIYTSSSIYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARVPLYGVSSGYYYYYGMDLWGPGTLVTVSS；

VL （SEQ ID NO:18）

DIVMTQTPASVSAAVGGTVTIKCRASQSISSYLAWYQQKPGQPPKRLIYKASTLASGVPSRFKGSASGTDFTLTISDLECADAATYYCQSNYGSSSSSYGWDNAFGGGTEVVVK；

VD mAb3：

VH （SEQ ID NO:27）

QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYSMGWVRQAPGEGLEYIGWINNGGTAYYASWAKGRFTISRTSTTVDLKMTSPTTEDTATYFCARYGISSGYYNLWGQGTLVTVSS；

VL （SEQ ID NO:28）

AQVLTQTPSSVSAAVGGTVTINCQASQSLYSNKNLAWYKQKPGQPPKLLIYWASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSGDCFAFGGGTEVVVK；

VD mAb4：

VH （SEQ ID NO:37）

QSVEESGGRLVTPGTPLTLTCTASGFSLSTYAMIWVRQAPGKGLEWIGIIHISGIIDYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARVYGLWGQGTLVTVSS；

VL（SEQ ID NO:38）

AQVLTQTASSVSAAVGGTVTISCQSSQSVYNNNWLAWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGSYDCSSGDCDGFGGGTEVVVK；

VD mAb5：

VH （SEQ ID NO:47）

QSVEESGGRLVTPGAPLTLTCTVSGFSLSTYAMSWVRQAPGKGPEWIGLIGSSGSTYYASWVNGRFTISKTSTTMDLKITGPTSEDTATYFCARGYSGDGWLYANNLWGQGTLVTVSS；

VL （SEQ ID NO:48）

DIVMTQTPASVEAAVGGTVTIKCQASQRISIYLSWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYGSSSSNYGNTFGGGTEVVVK；

VD mAb6：

VH （SEQ ID NO:57）

QSLEESGGRLVTPGGSLTLTCTVSGIDLSSYVMAWVRQAPGKGLEWIGIIYTDGSASYASWAKGRFTTSKTSTTVDLKITSPTTEDTATYFCARGIEEYTSSSYNLWGQGTLVTVSS；

VL（SEQ ID NO:58）

AQVLTQTPSPVSAAVGGTVTISCQSSQSVYNNNRLSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGYYSGYMNAFGGGTEVVVK；

VD mAb7：

VH （SEQ ID NO:67）

QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIGSSGDTYYASWAEGRFTASKTSTTVDLKITSPTTEDTATYFCAREHSYDDYGDYTGYFNLWGQGTLVTVSS；

VL（SEQ ID NO:68）

AIKMTQTPSSVSAAVGGTVTINCRASEDIESYLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQSAYYSSSALYNAFGGGTEVVVK；

VD mAb8：

VH （SEQ ID NO:77）

QSVEESGGRLVTPGTPLTLTCTASGFSLSSYAVSWVRQAPGKGLEWIGIISSSGITYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGNSYDDYLDIDWLDLWGQGTLVTVSS；

VL（SEQ ID NO:78）

AIKMTQTPSSVSAAVGGTVTINCRASEDIESLLAWYQQKPGQPPKLLIYDTSHLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQSAYDSNSPDTGAFGGGTEVVVK；

VD mAb9：

VH （SEQ ID NO:87）

QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIISVSGSTYYASWAEGRFTISKTSTTVDLKITSPTTEDTATYFCARGKYSYDDYGDNGDAFDPWGPGTLVTVSS；

VL（SEQ ID NO:88）

DVVMTQTPASVSAAVGGTVTINCQASESIYSNLAWYQQKPGQPPKLLIYAASYLASGVPSRFKGSGSGTEYTLTISGVQCADAATYYCQSADYSSSADNAFGGGTEVVVK；

VD mAb10：

VH （SEQ ID NO:97）

QSLEESGGRLVTPGTALTLTCTVSGFSLSSYAMNWVRQAPGKGLEWIGIIYSGGYASYASWAKGRVTISKTSTTVDLKITSPTTEDTATYFCARGDTGLNLWGQGTLVTVSS；

VL（SEQ ID NO:98）

AAVLTQTPSPVSAPVGGTVTISCQSSQSVYRNNWLSWYQQKPGQPPKLLIYLASTLASGVPNRFKGSGSGTQFTLTISSVQCDDAATYYCLGSYDDDPDNAFGGGTEVVVK；

VD mAb11：

VH （SEQ ID NO:107）

VL（SEQ ID NO:108）

AAVLTQTPSPVSVAVGGTVTINCQSSQSVDNNNYLSWYQQKPGQSPRLLIYDASNLASGVPDRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADTFVFGGGTEVVVK；

VD mAb12：

VH （SEQ ID NO:117）

VL（SEQ ID NO:118）

AAVLTQTPSPVSAAVGGTVTISCQSSQSVDNNNDLAWYQQKPGQPPKLLIYRSSKLASGVPSRFSGSGSGTQFSLTISGVQCDDAATYYCLGGYDDDAALNAFGGGTEVVVK；

VD mAb13：

VH （SEQ ID NO:127）

VL（SEQ ID NO:128）

QAVLTQTPSPVSAAVGGTVTISCQSSQSVYSNNRLSWFQQKPGQPPKLLIYYASTLASGVPSRFSGSGSGTQFTLTISGVQCDDGAIYYCLGGYDDDAETFAFGGGTEVVVK；

VD mAb14：

VH （SEQ ID NO:15）

QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGVTWVRQAPGKGLEWIGIIYTDGSTWYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAREGGYVDDADYFGLWGQGILVTVSS；

VL（SEQ ID NO:25）

AYDMTQTPASVEVTVGGTVTIKCQASQSIYNYLAWYQQKPGQPPKLLIYEASKLASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQGYTSGNVDNIFGGGTEVVVK；

VD mAb15：

VH （SEQ ID NO:105）

QSLEESGGRLVTPGTPLTLTCTASGFSLSTYYMTWVRQAPGEGLEYIGFITTGGSAYYASWVKGRFTISRTSTTVDLMIISPTTEDTATYFCARVVGCGYPCHYLSIWGQGTLVTVSS；

VL（SEQ ID NO:115）

DVVMTQTPASVEAAVGGTVTINCQASQSANSWLAWYQQKPGQPPKLLIYGTSNLESGVPSRFKGSGSGTQFTLTISDLECADAATYYCQCTYGIGDINVGLTFGGGTEVVVE。

Example 3: expression and purification of monoclonal antibodies against 25-OH-VD

(1) N293 suspension cells were cultured until the density reached (1-3). Times.10 ⁶ Cell transfection is carried out when the cell/ml and the survival rate is more than 80-90%;

(2) Light and heavy chain genes of 10 monoclonal antibodies VD mAb 1-VD mAb10 are synthesized according to the light and heavy chain gene sequences of the 10 monoclonal antibodies VD mAb 1-VD mAb10, and the synthesized light and heavy chain genes are respectively constructed into pcDNA3.4 plasmids; then, the recombinant plasmid containing the antibody light and heavy chain genes is transfected into N293 cells under the following conditions: the mass ratio PEI/plasmid was 3:1, transfected at a ratio of 2. Mu.g plasmid/1 ml N293 cells;

(3) Collecting cell supernatant 5-7 days after transfection, performing affinity purification by using Protein A resin to obtain high-purity monoclonal antibody, replacing elution buffer solution with PBS by dialysis, and measuring antibody concentration by using Nanodrop;

Next, the purity of the antibodies was checked by SDS-PAGE and HPLC as follows:

SDS-PAGE detection:

taking 2 mug of antibody to be detected, adding a proper amount of SDS-PAGE protein loading buffer to make the total volume be 20 mug; simultaneously preparing a reducing SDS-PAGE sample and a non-reducing SDS-PAGE sample, and then loading the samples; firstly, electrophoresis is carried out for 30min at 80V, and then electrophoresis is carried out at 120V until the separation of the strips is clear; and taking down the electrophoresis gel, performing coomassie brilliant blue dyeing, removing the dyeing liquid after 15min, washing with clear water, and then adding a decolorizing liquid for decolorizing until clear bands are seen.

The results are shown in FIG. 1, FIG. 1 showing: under reducing conditions, the 10 monoclonal antibodies all have two electrophoresis bands, and the corresponding molecular weights of the two electrophoresis bands are consistent with the sizes of the respective light chains and heavy chains; under non-reducing conditions, 10 monoclonal antibodies all have a single band, and the corresponding molecular weight of the electrophoretic band is consistent with the size of the respective intact antibody; these results indicate that the 10 monoclonal antibodies are expressed accurately.

In addition, all the electrophoresis bands were clear in edge and free of bands, indicating that the purity of the monoclonal antibodies obtained by the above-described expression and purification steps was high.

HPLC detection:

10. Mu.l of the purified antibody solution was taken and added to the inner tube, and the air bubbles were removed and put on the machine. The preparation HPLC uses 150mM phosphate buffer, the pH is adjusted to 7.0, and the buffer is subjected to vacuum filtration treatment for sterilization, and then the sterilization is placed in an ultrasonic instrument for ultrasonic degassing for standby. Starting the instrument, opening each module of the high performance liquid chromatograph, performing starting preheating treatment, firstly performing bubble removal treatment on an instrument system, then performing solution replacement by using a flowing opposite instrument, connecting the SEC column into the instrument, and balancing the column with a balancing buffer solution after bubble removal for later use. Loading: and opening software, performing methodological editing, generating a sequence, and then loading. Result analysis and processing: and opening the software, importing the result, analyzing and generating an analysis report.

HPLC results showed: the purity of the 10 monoclonal antibodies is more than 99 percent.

Example 4: antigen binding Activity and affinity detection of monoclonal antibodies against 25-OH-VD

In this example, 10 monoclonal antibodies VD mAb 1-VD mAb10 expressed and purified in example 3 were each subjected to antigen activity and affinity detection.

Binding Activity and affinity assays

(1) Antigen coating: diluting the antigen (i.e., the aforementioned conjugated protein 25-OHDD-BSA) to a suitable concentration, and then adding the diluted antigen to an empty ELISA plate in a volume of 100. Mu.l/well; placing the ELISA plate in a refrigerator at 4 ℃ and incubating overnight;

(2) closing: washing the plate for 5 times by using a plate washing machine, buckling, adding sealing liquid into the ELISA plate, placing 150 μl of sealing liquid into a 37 ℃ incubator, and sealing for 1h; washing the board for 5 times by a board washing machine, and buckling;

(3) incubating primary antibodies: diluting the antibody to be tested to a proper concentration gradient by using 1 XPBS; adding diluted antibody to be detected into an ELISA plate, wherein each hole is 100 mu l, and the reaction time is as follows: 37 ℃/1h; washing the board for 5 times by a board washing machine, and buckling;

(4) incubating a secondary antibody: the second enzyme-labeled antibody is diluted according to a proper proportion, then added into an enzyme-labeled plate, and the reaction time is 100 mu l per hole: 37 ℃/30min; washing the board for 5 times by a board washing machine, and buckling;

(5) color development: adding the color developing solution into the ELISA plate, wherein each hole is 100 mu l, and the reaction time is as follows: 37 degrees/15 min;

(6) termination and reading: adding a stop solution, wherein the volume is 100 mu l/hole; detection wavelength setting: the detection wavelength is 450nm, the reference wavelength is 620nm, and the detection result is derived into an Excel table.

(7) Data analysis: data analysis and mapping were performed using software, EC50 was fitted, and KD was deduced.

The results are shown in fig. 2, fig. 2 showing: the 10 monoclonal antibodies and antigen combined EC50 are less than 15ng/ml, KD reaches 10 ^-11 M。

As mentioned previously, for IgG antibodies, it is generally recognized in the art that when the KD for antigen binding reaches 10 ^-9 M is considered to have high affinity, and the KD value of the 10 monoclonal antibodies of the invention for binding with antigen reaches 10 ^-11 M is as much as 100 times the above defined value, thereby obtaining: the monoclonal antibody of the invention has extremely strong antigen binding activity and affinity.

Example 5: preparation of double-antibody sandwich method colloidal gold immunochromatography test strip based on anti-25-OH-VD monoclonal antibody

In this example, two colloidal gold immunochromatography test strips for detecting 25-OH-VD according to the principle of double-antibody sandwich method are prepared by using VD mAb1 and VD mAb2, VD mAb3 and VD mAb4 expressed and purified in example 3 as paired antibodies, respectively, and the schematic structure of the test strips is shown in FIG. 3; the preparation method of the test strip comprises the following steps:

(1) Preparation of Au-anti-25-OH-VD monoclonal antibody complex

(1) Taking 3ml of colloidal gold particles (colloid Jin Lijing: 20nm, maximum absorption peak wavelength of 529nm, absorbance of 2.0A), adding 18 μl of 0.1M K ₂ CO ₃ Uniformly mixing, adding 10 mug of an anti-25-OH VD antibody (namely a detection antibody), and reacting at normal temperature for 1 h;

(2) adding 600 μl of 5% BSA blocking solution into the system in the step (1), and blocking for 30min at normal temperature;

(3) after the completion of the blocking, the mixture was centrifuged at 11000rpm at 10℃for 15 minutes, the supernatant was discarded, and the precipitate was redissolved (i.e., concentrated 60-fold, 60X) with 50. Mu.l of a gold-labeled diluent to obtain an Au-anti-25-OH-VD monoclonal antibody complex; the gold standard diluent contains 100mM Tris buffer, pH7.5-8.5, 5% sucrose, 1% trehalose and 5% BSA.

(2) Gold drawing

(1) Closing line is marked: taking 5% BSA solution, setting the water yield of the instrument to be 3 mu L/cm, and placing the scratched bottom plate in a 37 ℃ oven for drying for 2 hours;

(2) diluting the final concentration of the Au-anti-25-OH-VD monoclonal antibody complex to 50X, setting the water yield to 2 mu L/cm, scribing at the position of a closed line, then placing a bottom plate in a baking oven at 37 ℃ for drying for 2 hours, and then placing a marking pad in an aluminum foil bag which is added with a drying agent in advance at the humidity of not more than 36%, sealing and preserving for later use;

(3) Coating quality control line C and detection line T

(1) Preparing a coating liquid of a quality control line C and a detection line T: the goat anti-rabbit antibody is diluted into 1.0mg/mL antibody solution by 10mM PBS buffer solution with pH of 7.4; another anti-25-OH-VD monoclonal antibody (i.e., capture antibody) was diluted to 1.0mg/mL with 10mM PBS buffer, pH 7.4;

(2) Coating: coating the coating liquid of the quality control line C and the detection line T to the positions of the quality control line and the detection line of the nitrocellulose membrane respectively, wherein the interval between C, T lines is 8mm, and the coating amount is 1 mu L/cm; placing the coated PVC bottom plate in a baking oven at 37 ℃ for 16 hours, then placing the PVC bottom plate in an aluminum foil bag with a desiccant added in advance under the humidity of not more than 36%, and sealing and preserving for later use;

(4) Pretreatment of sample pad

Cleaning a drying net, soaking and wetting a sample pad by using sample pad pretreatment liquid, draining by rotation until no water drops drip, then drying for 4 hours in a 37 ℃ oven, and then placing the sample pad into an aluminum foil bag which is pre-added with a drying agent at the humidity of not more than 36%, sealing and preserving for later use;

the sample pad pretreatment solution contains 10mM Tris buffer with pH of 8.5, 0.1% PVP 10, 0.1% PEG20000, 0.1% Tween20 and 0.1% sodium caseinate;

(5) Assembling and cutting film materials

Taking out the water absorption pad and the treated sample pad under the humidity of not higher than 36%, cutting according to the size of 10mm multiplied by 300mm, taking out the coated PVC base plate, and assembling according to the following procedures: firstly, attaching a sample pad on a PVC bottom plate, enabling a sample pad part to press a nitrocellulose membrane for 2mm, and finally attaching a water absorption pad, wherein the water absorption pad presses the nitrocellulose membrane for 2mm; cutting the assembled PVC bottom plate into 4mm to obtain the colloidal gold immunochromatographic test strip for detecting 25-OH-VD.

The antibody pairing mode of the test strip 1 is as follows: VD mAb1 (for preparing Au-antibody complex) +vd mAb2 (for T-line coating); the antibody pairing mode of the test strip 2 is as follows: VD mAb3 (for preparing Au-antibody complex) +vd mAb4 (for T-line coating).

Example 6: detection performance of double-antibody sandwich method colloidal gold immunochromatography test strip based on anti-25-OH-VD monoclonal antibody

In this example, the two colloidal gold immunochromatographic test strips prepared in example 5 were tested for their detection performance against 25-OH-VD in clinical serum samples and compared with the detection performance of the conventional Roche competition method.

Specifically, for the two colloidal gold immunochromatographic test strips prepared in example 5, a clinical serum sample is diluted by a sample diluent (the composition of which is 10mM BS+5% sodium dodecyl sulfate+5% EDTA disodium+0.5% guanidine isothiocyanate+0.1% procrin300, pH 7.0), the reaction is carried out for 10min at room temperature, then 75 mu l of sample is added, the sample is detected by a colloidal gold immunoassay instrument after 15 min, the detection value is read, and the data is processed to obtain the 25-OH-VD concentration in the sample of each dilution; for the Roche kit, the serum sample is diluted and then detected according to the specification flow, so that 25-OH-VD concentration data is obtained. And then, comparing the 25-OH-VD concentration data detected by the two test strips and the 25-OH-VD data detected by the Roche competition method with corresponding mass spectrometry assignment respectively, and preparing clinical correlation curves of three test methods (the two test strips and the Roche detection kit).

The results are shown in FIG. 4; wherein FIGS. 4A and 4B are graphs showing the results of clinical correlations (i.e., coincidence rate with "gold standard" mass spectrometry detection results) of the colloidal gold immunochromatographic test strip 1 and the test strip 2, respectively, the correlations of the two test strips being respectivelyR ² > 0.95 and R ² > 0.96; fig. 4C is a graph of results of the clinical relevance (i.e., coincidence rate with "gold standard" mass spectrometry detection results) of rogowski competition assay reagents, which relevance is: r is R ² =0.9。

The results show that compared with the traditional Roche competition method, the 25-OH-VD colloidal gold immunochromatography test strip prepared based on the paired antibody has obviously higher coincidence rate (more than 0.95) with the detection result of the gold standard mass spectrometry, so that the paired antibody has better clinical relevance.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region having amino acid sequences shown as SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 14, KAS and SEQ ID NO. 16, LCDR1, LCDR2 and LCDR3, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region, the amino acid sequence of which is shown as SEQ ID NO. 17; and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 18.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

a heavy chain with an amino acid sequence shown in SEQ ID NO. 19; and a light chain, the amino acid sequence of which is shown in SEQ ID NO. 20.

4. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', F (ab') ₂ Fd, fv, dAb, complementarity determining region fragments, single chain antibodies.

5. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that it is a human or chimeric antibody.

6. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that it is a bispecific or multispecific antibody.

7. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. A nucleic acid construct comprising the polynucleotide of claim 7, and at least one expression regulatory element operably linked to the polynucleotide.

9. A recombinant vector comprising the polynucleotide of claim 7, or the nucleic acid construct of claim 8.

10. A transformed host cell comprising the polynucleotide of claim 7, the nucleic acid construct of claim 8, or the recombinant vector of claim 9.

11. Use of a monoclonal antibody or antigen binding fragment thereof according to any one of claims 1-6, a polynucleotide according to claim 7, a nucleic acid construct according to claim 8, a recombinant vector according to claim 9 and/or a transformed host cell according to claim 10 for the preparation of a detection reagent or kit for detecting 25-hydroxyvitamin D.

12. Use of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 in combination with another monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D for the preparation of a detection reagent or kit for detecting 25-hydroxyvitamin D, wherein the other monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D comprises:

a heavy chain variable region having amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 4, LAS and SEQ ID NO. 6, LCDR1, LCDR2 and LCDR3, respectively.

13. The use according to claim 12, wherein the further monoclonal antibody or antigen binding fragment thereof directed against 25-hydroxyvitamin D comprises:

a heavy chain variable region, the amino acid sequence of which is shown as SEQ ID NO. 7; and a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO. 8.

14. The use according to claim 13, wherein the further monoclonal antibody or antigen binding fragment thereof directed against 25-hydroxyvitamin D comprises:

The heavy chain has an amino acid sequence shown in SEQ ID NO. 9; and a light chain, the amino acid sequence of which is shown in SEQ ID NO. 10.

15. A detection reagent for detecting 25-hydroxyvitamin D comprising the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-6, the polynucleotide of claim 7, the nucleic acid construct of claim 8, the recombinant vector of claim 9, and/or the transformed host cell of claim 10.

16. The detection reagent according to claim 15, wherein the detection reagent is a double antibody sandwich immunoassay kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and another monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D comprising:

17. The detection reagent according to claim 16, wherein the other monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D comprises:

18. The detection reagent according to claim 17, wherein the other monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D comprises:

19. The detection reagent according to any one of claims 16 to 18, wherein the double-antibody sandwich immunoassay kit comprises a double-antibody sandwich immunochromatographic test strip in which:

a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 as a capture antibody and the other monoclonal antibody or antigen-binding fragment thereof directed against 25-hydroxyvitamin D as a detection antibody.

20. A method of making the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, the method comprising: allowing the transformed host cell of claim 10 to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.