WO2024140618A1 - 1,3-β-D-GLUCAN BINDING PROTEIN, PREPARATION METHOD, AND USE - Google Patents

Abstract

The present invention relates to the technical field of in-vitro diagnosis, and in particular to a 1,3-β-D-glucan binding protein, a preparation method, and a use. The antifungal 1,3-β-D-glucan binding protein provided by the present invention has high specificity; a monoclonal antibody is a gene expression product; after three complementary determining regions (CDRs) of a wild-type binding protein are determined, all non-alanine amino acids in the CDRs are further subjected to point mutation one by one to form alanine; and by means of comparison of changes in binding forces of different mutants and a substrate, an antibody in which the binding force of the substrate is 100 times the binding force of the wild-type binding protein is obtained on the basis of the wild-type binding protein. The binding protein is applied to a chemiluminescence method, such that the detection threshold is greatly widened, the detection lower limit is reduced, and the quantitative sensitivity is enhanced. The antibody is obtained by artificial mutation, has the advantage of high stability, and is also immune to HAMA interference.

Description

1,3-β-D-glucan binding protein, preparation method and application

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure claims the priority of Chinese patent application number CN202211737487.2, filed with the Chinese Patent Office on December 30, 2022, and entitled “1,3-β-D-glucan binding protein, preparation method and application”, the entire contents of which are incorporated by reference in this disclosure.

Technical Field

The present disclosure relates to the field of in vitro diagnostic technology, and in particular to 1,3-β-D-glucan binding protein, a preparation method and application thereof.

Background technique

At present, the commonly used fungal 1,3-β-D-glucan detection technology is the horseshoe crab reagent method, and the detection time is about 50 minutes. The key raw material of the horseshoe crab reagent comes from the national second-level wild protected animal: horseshoe crab, and the horseshoe crab reagent is extracted from horseshoe crab blood. However, the growth cycle of horseshoe crabs is long and it is extremely difficult to breed. They can only be collected by catching wild horseshoe crabs, and blood can be collected from each horseshoe crab. About 100 to 300 mL of blood can be collected. After several years of hunting, horseshoe crabs have become an endangered species, and there is a risk of insufficient supply of raw materials for horseshoe crab reagents. In addition, the horseshoe crab reagent prepared from natural horseshoe crab blood has a large batch difference, which leads to increased product production costs and poor reproducibility. The detection principle of the horseshoe crab reagent method is: 1,3-β-D-glucan can specifically activate the G factor in the horseshoe crab reagent, and then activate procoagulant to form coagulase, and coagulase catalyzes the subsequent color reaction or turbidity reaction. Taking the fungal 1,3-β-D-glucan detection product of Dana (Tianjin) Biotechnology Co., Ltd. as an example, the kit uses the horseshoe crab reagent colorimetric method. The sample is first pretreated for 10 minutes, and then the horseshoe crab reagent and color substrate are added for incubation for 40 minutes. 1,3-β-D-glucan can specifically activate the G factor in the reaction main agent, and then activate the procoagulant. The coagulase hydrolyzes the color substrate in the reaction to produce free p-nitroaniline (pNA), thereby causing the absorbance change. The concentration of 1,3-β-D-glucan is quantified according to the absorbance change rate of the dynamic detection solution. The total time of this method is about 50 minutes, and the linear lower limit is 37.5pg/mL.

However, horseshoe crab reagent can also activate procoagulant to form coagulase under the action of bacterial endotoxins, and bacterial endotoxins are widely present in nature, so horseshoe crab reagent is easily interfered by bacterial endotoxins.

In the method of replacing horseshoe crab reagent to detect 1,3-β-D-glucan, the antibodies used have limited binding ability with the substrate, have not undergone systematic modification, and mostly retain natural properties. Taking Roche's PCT detection product as an example, the reagent adopts the sandwich method, adding samples, biotin-labeled antibodies and ruthenium complex-labeled antibodies to the reaction cup, and incubating for a period of time to form an antigen-antibody sandwich complex. Add magnetic beads coated with streptavidin for incubation, and the complex binds to the magnetic beads through the action of biotin and streptavidin. The reaction solution is sucked into the measuring cell, and the magnetic beads are adsorbed on the electrode surface by electromagnetic action. The substances not bound to the magnetic beads are removed by ProCell, and a certain voltage is applied to the electrode to make the complex chemiluminescent, and the luminescence intensity is measured by a photomultiplier. The luminescence value is positively correlated with the concentration of the analyte in the sample. The total time of this method is about 18 minutes, and the detection limit is low. It is mainly limited by the ability of the antibody to bind to the substrate, which directly affects its sensitivity and specificity in clinical applications. Therefore, more excellent detection reagents and detection methods are urgently needed.

Summary of the invention

The present disclosure provides a binding protein comprising a 1,3-β-D-glucan binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequences, or has at least 80% sequence identity with the complementarity determining region of the following amino acid sequences and has a binding affinity to 1,3-β-D-glucan of KD≤10 ^-9 mol/L;

The complementary determining region CDR-VH1 is X1-X2-X3-W-X4-X5, wherein,

X1 is N or A, X2 is D or A, X3 is F or A, X4 is I or A, and X5 is C or A;

The complementary determining region CDR-VH2 is X1-X2-V-X3-D-X4-X5-X6-F-G-F-S-A-S-X7-A-K-G, wherein,

X1 is C or A, X2 is M or A, X3 is P or A, X4 is G or A, X5 is S or A, X6 is G or A, X7 is W or A;

The complementarity determining region CDR-VH3 is Y-X1-X2-V-X3-G-P-Y-S-X4-X5-X6, wherein

X1 is G or A, X2 is D or A, X3 is G or A, X4 is F or A, X5 is K or A, X6 is I or A;

The complementary determining region CDR-VL1 is Q-X1-X2-X3-X4-X5-G-Y-X6-N-N-X7-A, wherein

X1 is S or A, X2 is S or A, X3 is Q or A, X4 is S or A, X5 is V or A, X6 is G or A, X7 is L or A;

The complementary determining region CDR-VL2 is X1-A-S-R-L-A-S, wherein,

X1 is G or A;

The complementary determining region CDR-VL3 is A-G-X1-Y-X2-I-I-T-X3-X4-C-V-X5, wherein,

X1 is D or A, X2 is G or A, X3 is D or A, X4 is T or A, and X5 is F or A.

Optionally, in the complementary determining region CDR-VH1, X3 is A;

Alternatively, in the complementary determining region CDR-VH2, X3 is A;

Alternatively, in the complementary determining region CDR-VH3, X4 is A;

Alternatively, in the complementary determining region CDR-VL1, X2 is A;

Alternatively, in the complementary determining region CDR-VL2, X1 is A;

Alternatively, in the complementarity determining region CDR-VL3, X4 is A.

Optionally, the complementarity determining region comprises any combination of the following (a) to (i):

(a) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL1, X1 is A;

(b) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL2, X1 is A;

(c) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL3, X1 is A;

(d) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL3, X4 is A;

(e) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL1, X1 is A;

(f) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL2, X1 is A;

(g) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL3, X1 is A;

(h) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL3, X4 is A;

(i) in the complementary determining region CDR-VH3, X1 is A and X4 is A; in the complementary determining region CDR-VL1, X1 is A; in the complementary determining region CDR-VL2, X1 is A; in the complementary determining region CDR-VL3, X1 is A and X4 is A.

Optionally, the binding protein comprises at least 3 CDRs; or, the binding protein comprises 6 CDRs;

Optionally, the binding protein is one of nanoantibody, F(ab')2, Fab', Fab, Fv, scFv, bispecific antibody and antibody minimum recognition unit.

Optionally, the binding protein comprises heavy chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 whose sequences are sequentially shown in SEQ ID NOs: 1 to 4, and/or light chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 whose sequences are sequentially shown in SEQ ID NOs: 5 to 8;

Optionally, the binding protein further comprises an antibody constant region sequence;

Optionally, the constant region sequence is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD;

Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human;

Optionally, the constant region is derived from rabbit.

The present disclosure also provides a biomaterial, the biomaterial comprising any one of the following:

(a) a nucleic acid molecule encoding the binding protein described in any one of the above;

(b) a vector comprising the nucleic acid molecule described in (a);

(c) a host cell comprising the nucleic acid molecule of (a) or the vector of (b).

The present disclosure also provides a method for preparing the binding protein described in any one of the above items, the preparation method comprising culturing the host cells, and recovering the produced binding protein from the culture medium or from the cultured host cells.

The present disclosure also provides use of any of the binding proteins described above in the preparation of a diagnostic agent, a test strip or a kit for diagnosing fungal infection.

The present disclosure also provides a 1,3-β-D-glucan detection test strip, wherein the detection line of the test strip is obtained by streaking the binding protein described in any one of the above items.

The present disclosure also provides a reagent or a kit for detecting fungal 1,3-β-D-glucan, wherein the reagent or the kit comprises the binding protein described in any one of the above items.

The present disclosure also provides the use of the binding protein described in any one of the above, or the 1,3-β-D-glucan detection test strip, or the reagent or kit for detecting fungal 1,3-β-D-glucan, for diagnosing fungal infection.

The present disclosure also provides a 1,3-β-D-glucan detection method for non-diagnostic purposes, wherein the detection method comprises chemiluminescence detection, wherein the chemiluminescence detection comprises a sample pretreatment step, wherein the pretreatment step comprises any one of (a) to (c):

(a) Treat the sample with alkali solution at 37°C;

(b) The sample was treated with EDTA-2Na solution at 100°C;

(c) treating the sample to be tested with the acidic sample release solution at 2 to 40° C.;

Alternatively, the detection method includes detection using the test strip;

The 1,3-β-D-glucan source includes Candida albicans or Aspergillus fumigatus.

The present disclosure also provides a method for detecting fungi, comprising:

A) contacting the binding protein described in any one of the above, or the 1,3-β-D-glucan detection test strip, or the reagent or kit for detecting fungal 1,3-β-D-glucan with a sample from the subject under conditions sufficient for a binding reaction to perform a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The present disclosure also provides a method for diagnosing a subject in a fungal infection or a disease associated with a fungal infection, comprising:

A) contacting the binding protein described in any one of the above, or the 1,3-β-D-glucan detection test strip, or the reagent or kit for detecting fungal 1,3-β-D-glucan with a sample from the subject under conditions sufficient for a binding reaction to perform a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

FIG1 is a graph showing the effect of each point mutation of the heavy chain on binding ability in Example 3 of the present disclosure;

FIG2 is a graph showing the effect of each point mutation of the light chain on binding ability in Example 3 of the present disclosure;

FIG3 is a pull-down diagram of the 3D structure of the wild-type WT antibody in Example 4 of the present disclosure;

Figure 4 is the heavy chain SAVES Verify 3D scoring result of the wild-type WT antibody in Example 4 of the present disclosure;

Figure 5 is the light chain SAVES Verify 3D scoring result of the wild-type WT antibody in Example 4 of the present disclosure;

FIG6 is a comparison diagram of the heavy chain docking results and experimental results in Example 4 of the present disclosure;

FIG7 is a comparison diagram of the light chain docking results and experimental results in Example 4 of the present disclosure;

FIG8 is a schematic diagram of the binding between the wild-type antibody and the antigen obtained in Example 4 of the present disclosure;

FIG9 is a linear result of wild-type antibody detection obtained in Example 5 of the present disclosure;

FIG. 10 is a linear result of mutant antibody detection obtained in Example 5 of the present disclosure.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments.

Therefore, the following detailed description of the embodiments of the present disclosure provided in the accompanying drawings is not intended to limit the scope of the present disclosure claimed for protection, but merely represents selected embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and/or". In addition, the use of the term "including" and other forms is non-limiting.

Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques disclosed herein are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly implemented in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

In order that the present disclosure may be more readily understood, selected terms are defined below.

The term "amino acid" refers to a naturally occurring or non-naturally occurring carboxyl α-amino acid. The term "amino acid" as used in the present application may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: A1a, single letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, c), glutamine (G1n, Q), glutamic acid (G1u, E), glycine (G1y, G), histidine (His, H), isoleucine (I1e, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Non-naturally occurring amino acids include, but are not limited to, α-aminoadipic acid, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

The term "binding protein comprising a 1,3-β-D-glucan binding domain" generally refers to a protein or protein fragment that can specifically bind to 1,3-β-D-glucan and comprises a CDR region.

The term "antibody", or immunoglobulin, includes polyclonal antibodies, monoclonal antibodies, and antigen compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. In addition, "antibodies" include naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, as well as related synthetic isoforms.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework regions of an antibody, i.e., the framework regions that make up the combination of the light and heavy chains, serve to position and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework" or "FR" regions refer to the contiguous regions (FR1, FR2, FR3 and FR4) into which each antibody variable domain framework is further subdivided into CDRs.

Typically, the variable regions VL/VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The present disclosure provides a binding protein for antifungal 1,3-β-D-glucan with high specificity. It is expected that the binding protein has high binding affinity with 1,3-β-D-glucan and can be used in detection such as chemiluminescence or test strip method. It is expected that the detection threshold can be greatly broadened, the detection limit can be reduced, the quantitative sensitivity can be enhanced, and the influence of HAMA interference can be avoided.

Some embodiments of the present disclosure provide a binding protein comprising a 1,3-β-D-glucan binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequences, or has at least 80% sequence identity with the complementarity determining region of the following amino acid sequences and has a binding affinity with 1,3-β-D-glucan of KD≤10 ^-9 mol/L;

The complementary determining region CDR-VH1 is X1-X2-X3-W-X4-X5, wherein,

X1 is N or A, X2 is D or A, X3 is F or A, X4 is I or A, and X5 is C or A;

The complementary determining region CDR-VH2 is X1-X2-V-X3-D-X4-X5-X6-F-G-F-S-A-S-X7-A-K-G, wherein,

X1 is C or A, X2 is M or A, X3 is P or A, X4 is G or A, X5 is S or A, X6 is G or A, X7 is W or A;

The complementarity determining region CDR-VH3 is Y-X1-X2-V-X3-G-P-Y-S-X4-X5-X6, wherein

X1 is G or A, X2 is D or A, X3 is G or A, X4 is F or A, X5 is K or A, X6 is I or A;

The complementary determining region CDR-VL1 is Q-X1-X2-X3-X4-X5-G-Y-X6-N-N-X7-A, wherein

X1 is S or A, X2 is S or A, X3 is Q or A, X4 is S or A, X5 is V or A, X6 is G or A, X7 is L or A;

The complementary determining region CDR-VL2 is X1-A-S-R-L-A-S, wherein,

X1 is G or A;

The complementary determining region CDR-VL3 is A-G-X1-Y-X2-I-I-T-X3-X4-C-V-X5, wherein,

X1 is D or A, X2 is G or A, X3 is D or A, X4 is T or A, and X5 is F or A.

In an optional embodiment, in the complementary determining region CDR-VH1, X3 is A;

In the complementary determining region CDR-VH1, X3 is A;

Alternatively, in the complementary determining region CDR-VH2, X3 is A;

Alternatively, in the complementary determining region CDR-VH3, X4 is A;

Alternatively, in the complementary determining region CDR-VL1, X2 is A;

Alternatively, in the complementary determining region CDR-VL2, X1 is A;

Alternatively, in the complementarity determining region CDR-VL3, X4 is A.

In an optional embodiment, the complementarity determining region includes any combination of the following (a) to (i):

(a) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL1, X1 is A;

(b) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL2, X1 is A;

(c) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL3, X1 is A;

(d) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL3, X4 is A;

(e) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL1, X1 is A;

(f) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL2, X1 is A;

(g) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL3, X1 is A;

(h) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL3, X4 is A;

(i) in the complementary determining region CDR-VH3, X1 is A and X4 is A; in the complementary determining region CDR-VL1, X1 is A; in the complementary determining region CDR-VL2, X1 is A; in the complementary determining region CDR-VL3, X1 is A and X4 is A.

In an optional embodiment, the binding protein comprises at least 3 CDRs; or, the binding protein comprises 6 CDRs.

It is well known in the art that the binding specificity and affinity of an antibody are mainly determined by the CDR sequence. According to mature and well-known existing technologies, the amino acid sequence of the non-CDR region can be easily changed to obtain a variant with similar biological activity. Therefore, the present disclosure also includes "functional derivatives" of the binding protein. "Functional derivatives" refer to variants obtained by deletion, substitution or insertion of one or more amino acid residues. A functional derivative retains the activity of an antibody that can bind to 1,3-β-D-glucan. "Functional derivatives" may include "variants" and "fragments" because they have exactly the same CDR sequence as the binding protein described in the present disclosure and therefore have similar biological activities.

In an optional embodiment, the antigen binding domain has at least 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity with the complementarity determining region of the following amino acid sequence and has a binding affinity with 1,3-β-D-glucan of ^KD≤10-9 mol/L. The KD value can also be selected as ^10-8 mol/L, ^10-7 mol/L, etc.

Optionally, the binding protein is one of nanoantibody, F(ab')2, Fab', Fab, Fv, scFv, bispecific antibody and antibody minimum recognition unit.

In an optional embodiment, the binding protein includes heavy chain backbone regions FR-H1, FR-H2, FR-H3 and FR-H4 whose sequences are shown in SEQ ID NO: 1 to 4, and/or light chain backbone regions FR-L1, FR-L2, FR-L3 and FR-L4 whose sequences are shown in SEQ ID NO: 5 to 8 (Table 1).

Table 1

In an alternative embodiment, the binding protein further comprises an antibody constant region sequence.

In an optional embodiment, the constant region sequence is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In an alternative embodiment, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

In an alternative embodiment, the constant region is derived from rabbit.

In an optional embodiment, the amino acid sequence of the constant region of the heavy chain is GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:9).

In an alternative embodiment, the amino acid sequence of the constant region of the light chain is GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 10).

Some embodiments of the present disclosure provide a biomaterial, wherein the biomaterial comprises any one of the following:

(a) a nucleic acid molecule encoding the binding protein described in any one of the above;

(b) a vector comprising the nucleic acid molecule described in (a);

(c) a host cell comprising the nucleic acid molecule of (a) or the vector of (b).

The nucleic acid molecules include conservatively substituted variants thereof (eg, substitutions of degenerate codons) and complementary sequences. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and fragments thereof such as oligonucleotides.

In the vector, the nucleic acid sequence therein is operably linked to at least one regulatory sequence. "Operably linked" means that the coding sequence is linked to the regulatory sequence in a manner that allows the expression of the coding sequence. The regulatory sequence is selected to direct the expression of the target protein in a suitable host cell and includes a promoter, an enhancer and other expression control elements.

The vector may refer to a molecule or reagent that contains a nucleic acid molecule or fragment thereof disclosed herein, can carry genetic information, and can deliver genetic information to a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids, and minichromosomes. A vector may be a cloning vector (i.e., a vector for transferring genetic information to a cell, which can propagate the cell and can select the cell with or without the genetic information) or an expression vector (i.e., a vector that contains necessary genetic elements to allow the genetic information of the vector to be expressed in a cell). Therefore, a cloning vector may contain a selection marker, and a replication origin that matches the cell type specified by the cloning vector, while an expression vector contains regulatory elements necessary for affecting expression in a specified target cell.

The nucleic acid molecules or fragments thereof disclosed herein can be inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid fragments disclosed herein, and this new vector is also a part of the present disclosure. The vector may include a plasmid, a phage, a cosmid, a minichromosome or a virus, and also includes naked DNA that is transiently expressed only in specific cells. The cloning vector and expression vector disclosed herein can replicate spontaneously, and therefore can provide a high copy number for the purpose of high-level expression or high-level replication for subsequent cloning. The expression vector may include a promoter for driving the expression of the nucleic acid fragments disclosed herein, an optional nucleic acid sequence encoding a signal peptide that secretes or integrates the peptide expression product into the membrane, the nucleic acid fragments disclosed herein, and an optional nucleic acid sequence encoding a terminator. When the expression vector is operated in a production strain or cell line, the vector may be integrated into the genome of the host cell when introduced into the host cell, or it may not be integrated into the genome of the host cell. The vector usually carries a replication site, and a marker sequence that can provide phenotypic selection in the transformed cell.

The expression vector of the present disclosure is used to transform host cells. Such transformed cells are also a part of the present disclosure, and can be cultured cells or cell lines for propagating nucleic acid fragments and vectors of the present disclosure, or for recombinant preparation of polypeptides of the present disclosure. Transformed cells of the present disclosure include microorganisms such as bacteria (such as Escherichia coli, Bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, and optionally, host cells are from mammalian cells, such as CHO cells. The transformed cells can replicate nucleic acid fragments of the present disclosure. When recombinant preparation of peptide combinations of the present disclosure, the expression products can be exported to the culture medium or carried on the surface of the transformed cells.

Some embodiments of the present disclosure provide a method for preparing the binding protein described in any one of the above items, the preparation method comprising culturing the host cells described in the second aspect, and recovering the produced binding protein from the culture medium or from the cultured host cells.

The preparation method can be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under appropriate conditions to express the binding protein. The host cell can also be transfected with one or more expression vectors, which can contain DNA encoding at least a portion of the binding protein alone or in combination. The binding protein can be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (such as ion exchange, gel filtration, affinity chromatography, etc.) and/or electrophoresis.

Construction of suitable vectors containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques known in the art. Isolated plasmids, DNA sequences or synthetic oligonucleotides are cut, tailed and re-ligated as desired. Mutations can be introduced into the coding sequence by any method to produce variants of the present disclosure, and these mutations can include deletions or insertions or substitutions, etc.

Some embodiments of the present disclosure provide use of any of the binding proteins described above in the preparation of a diagnostic agent, a test strip or a kit for diagnosing fungal infection.

Some embodiments of the present disclosure provide a 1,3-β-D-glucan detection test strip, wherein the detection line of the test strip is obtained by streaking the binding protein described in any one of the above items.

Some embodiments of the present disclosure provide a reagent or kit for detecting fungal 1,3-β-D-glucan, wherein the reagent or the kit comprises the binding protein described above.

Some embodiments of the present disclosure provide the binding protein described above, or the 1,3-β-D-glucan detection test strip, or the reagent or kit for detecting fungal 1,3-β-D-glucan, for use in diagnosing fungal infection.

Some embodiments of the present disclosure provide a method for detecting 1,3-β-D-glucan for non-diagnostic purposes, wherein the detection method comprises chemiluminescent detection, wherein the chemiluminescent detection comprises a pretreatment step of a sample to be tested, wherein the pretreatment step comprises any one of (a) to (c):

(a) Treat the sample with alkali solution at 37°C;

(b) The sample was treated with EDTA-2Na solution at 100°C;

(c) treating the sample to be tested with the acidic sample release solution at 2 to 40° C.;

Alternatively, the detection method comprises detection using the test strip described in the fifth aspect;

The 1,3-β-D-glucan source includes Candida albicans or Aspergillus fumigatus.

In addition, the present disclosure also provides a method for detecting 1,3-β-D-glucan in a sample to be tested, comprising:

(a) contacting 1,3-β-D-glucan in the test sample with any one of the binding proteins described above to form an immune complex; and

(b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the 1,3-β-D-glucan in the test sample;

The binding protein may be labeled with an indicator that shows signal intensity, so that the complex can be easily detected.

Optionally, in step (a), the immune complex further comprises a second antibody, and the second antibody binds to the binding protein;

Optionally, the binding protein forms a paired antibody with the second antibody in the form of a first antibody, which is used to bind to different antigenic epitopes of 1,3-β-D-glucan;

The secondary antibody may be labeled with an indicator that shows signal intensity, so that the complex can be easily detected.

Optionally, in step (a), the immune complex further comprises a second antibody, and the second antibody binds to the 1,3-β-D-glucan;

Wherein, the binding protein serves as the antigen of the second antibody, and the second antibody can be labeled with an indicator showing signal intensity so that the complex can be easily detected.

Optionally, the indicator showing signal intensity includes any one of fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal gold or enzymes.

Optionally, the fluorescent substance includes Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX 、5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein、5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein、5-carboxyfluorescein、5-carboxyrhodamine、6-carboxyrhodamine、6-carboxytetramethylrhodamine、Cascade Blue、Cy2、Cy3、Cy5、Cy7、6-FAM、dansyl chloride、fluorescein、HEX、6-JOE、NBD(7-nitrobenzo-2- any of phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, trisbipyridyldiamine europium, europium cryptates or chelates, diamines, bicyanines, La Jolla blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, or Texas Red.

Optionally, the radioactive isotope includes any one of ¹¹⁰ In, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ⁹⁴ mTc, ⁹⁴ Tc, ⁹⁹ mTc, ¹²⁰ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ^154-158 Gd, ³² P, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁵¹ Mn, ⁵² mMn, ⁵⁵ Co, ⁷² As, ⁷⁵ Br, ⁷⁶ Br, ⁸² mRb or ⁸³ Sr.

Optionally, the enzyme includes any one of horseradish peroxidase, alkaline phosphatase and glucose oxidase.

Optionally, the fluorescent microspheres are polystyrene fluorescent microspheres, which contain rare earth fluorescent ions europium.

Some embodiments of the present disclosure also provide a method for detecting fungi, comprising:

A) contacting the binding protein described in any one of the above, or the 1,3-β-D-glucan detection test strip, or the reagent or kit for detecting fungal 1,3-β-D-glucan with a sample from the subject under conditions sufficient for a binding reaction to perform a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

Some embodiments of the present disclosure also provide a method for diagnosing fungal infection or a disease associated with fungal infection in a subject, comprising:

A) contacting the binding protein described in any one of the above, or the 1,3-β-D-glucan detection test strip, or the reagent or kit for detecting fungal 1,3-β-D-glucan with a sample from the subject under conditions sufficient for a binding reaction to perform a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The fungus is a fungus containing 1,3-β-D-glucan. Optionally, the fungus includes Candida albicans or Aspergillus fumigatus.

The antifungal 1,3-β-D-glucan binding protein provided by the present disclosure has high specificity. The monoclonal antibody is a gene expression product. After determining the three complementarity determining regions (CDRs) of the wild-type binding protein, the present disclosure further mutates all non-alanine amino acids in the CDR region to alanine one by one. By comparing the changes in the binding force between different mutants and substrates, an antibody with a substrate binding force 100 times that of the wild-type binding protein is obtained on the basis of the wild-type binding protein. When applied in the chemiluminescence method, its detection threshold is greatly broadened, its detection limit is reduced, and its quantitative sensitivity is enhanced. The antibody is obtained by artificial mutation, has the advantages of high stability, small batch difference, and is not affected by cell line degeneration. At the same time, different animal-derived vectors can be selected for expression, which greatly improves the antibody production yield, reduces production costs, and avoids the interference of HAMA.

Example

In conjunction with the accompanying drawings, some embodiments of the present disclosure are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

Example 1 Fungal 1,3-β-D-glucan immunization experiment rabbit

The experimental rabbits (50 μg/rabbit) were subcutaneously immunized with fungal 1,3-β-D-glucan (Shanghai Pumai Biotechnology Co., Ltd., catalog number: E-BGOSAG) coupled with BSA or KLH. The first immunization was performed with fungal 1,3-β-D-glucan emulsified with complete Freund's adjuvant, and then the second, third, fourth, fifth, sixth, and seventh immunizations were performed with fungal 1,3-β-D-glucan emulsified with incomplete Freund's adjuvant every other week. After the seventh immunization, pure antigen was injected into the ear vein for booster immunization, rabbit blood was collected, and the titer of the gradient diluted serum was determined by ELISA to screen rabbits with specific antibody titers exceeding 500,000 in the serum. The steps of ELISA determination were: 1,3-β-D-glucan 0.2 μg/ml, 100 μL/well, ELISA plate coated at 80℃, 5% skim milk powder, 200 μL/well, blocked at 37℃ for 2h, the blocking solution was discarded, and dried at 37℃ for use. Rabbit blood was collected at 3000 rpm, and serum was collected after centrifugation. Serum was diluted from 1:10000 to 1:1280000 with PBS and added to the ELISA plate at 100 μL/well at 37°C for 1 hour. The reaction solution was discarded, patted dry, and washed 3 times with PBST. Sheep anti-rabbit secondary antibody (Beijing Solebow Technology Co., Ltd., catalog number: SE134) diluted 1:6000 with PBS was added at 100 μL/well at 37°C for 45 minutes. The reaction solution was discarded, patted dry, and washed 5 times with PBST. 100 μL TMB was added at 37°C for 10 minutes. The reaction solution was discarded, patted dry, and washed 5 times with PBST. 100 μL TMB was added at 37°C for 10 minutes. The rabbit serum titer test results are shown in Table 2 below:

Table 2

Example 2 Preparation of antifungal 1,3-β-D-glucan antibodies

On the basis of Example 1, three days before fusion, rabbits with an immune titer exceeding 500,000 were boosted with the same inoculation volume as the previous immunization, without adjuvant, and injected into the ear vein. One day before fusion, feeder cells were prepared, 10 mL of HAT selection culture medium was drawn with a sterile syringe and injected into the rabbit's abdominal cavity, the abdomen was gently rubbed with an alcohol cotton ball, and the culture medium was withdrawn. Add 40 mL of HAT culture medium, spread into 4 96-well cell culture plates, 100 μL/well, 37°C, 5% CO ₂ , and cultured in a cell culture incubator. One week before fusion, myeloma cells (Sp2/0 cells) were revived and cultured with PRMI-1640 culture medium containing 10% fetal bovine serum, and subcultured in a 37°C, 5% CO ₂ incubator. Cells in the logarithmic growth phase were collected into a centrifuge tube, the cells were counted, and the cells were diluted to 10 ⁷ /ml for standby use. Take the rabbit that was boosted for 3 days and remove the spleen aseptically on the clean bench, wash it several times in a sterile plate, and peel off the connective tissue. Place the spleen on a microporous copper mesh and add fresh RPMI-1640 culture medium. First, use a syringe to draw the culture medium and inject it from one section of the spleen to blow off the spleen cells. After repeating several times, use the inner stopper of the syringe to gently grind the remaining spleen until there is no obvious red tissue mass. Gently blow the spleen cell suspension in the dish and transfer it to a 50mL centrifuge tube. Centrifuge at 1000r/min for 5min, collect the spleen cells, count and set aside. Mix the immune rabbit spleen cells and Sp2/0 cells at a cell count of 10:1, add them to a 50mL centrifuge tube, centrifuge at 1000r/min for 5min, discard the supernatant, and gently rub in the palm of your hand to fully mix the two cells. Place the centrifuge tube in a 100mL blue-capped bottle filled with 37°C hot water. Add the preheated 1mL DMSO/PEG dropwise into the fusion tube within 1min, slowly at first and then quickly. Gently rotate the centrifuge tube while adding. Then immediately add RPMI-1640 culture medium without antibodies and blood to terminate the reaction. Add 1 mL in the first minute, 2 mL in the second minute, 3 mL in the third minute, and 4 mL in the fourth minute. Incubate at 37°C in a water bath for 5 minutes, then centrifuge at 800r/min for 5 minutes, discard the supernatant, suspend the precipitate with HAT, mix it in 40 mL of HAT selection culture medium containing 20% calf serum preheated at 37°C, and spread it into a 96-well cell plate with feeder cells, 100 μL/well, and place the culture plate in a 37°C, 5% CO ₂ incubator for culture. After 7 days, half of the cell plate will be replaced with fresh HAT culture medium, and after 10 days, the medium will be fully replaced with HT culture medium. The cells tested positive in the 96-well plate were subcloned by limiting dilution method: first, feeder cells were prepared according to the above method, hybridoma cells to be cloned were counted, cells were diluted to 5-8 cells/ml with HT culture medium, and added to 100μL/well of the 96-well cell plate with feeder cells. One 96-well cell plate was used for each hybridoma cell clone, and cultured in a cell culture incubator at 37℃ and 5% CO _2. After about 5 days, the number of clones in the cell wells was counted and marked. After 7 days, new culture medium was replaced and the cells were tested when 1/3 to 1/2 of the bottom of the well was covered. After 2-3 cloning, when all the cell wells of the 96-well plate were positive, the culture could be expanded, fixed and frozen. The hybridoma cells that were positive and fixed were expanded and frozen. The process was as follows: the hybridoma cells that were growing vigorously and in good condition were gently blown off the cell bottle with anti-blood-free DMEM, centrifuged at 1000r/min for 5 minutes, and the supernatant was discarded. Add freezing solution (containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, 10% DMSO), blow the cells apart and divide them into cell cryopreservation tubes. Place the cryopreservation tubes in a freezing box at -70°C refrigerator, and transfer the cryopreservation tubes into liquid nitrogen one day later. After cloning the genes of specific antibodies from these cell lines, construct a recombinant expression vector pCMVp-NEO-BAN-Ab containing the specific antibody gene, transform the recombinant expression vector into CHO cells for expression, purify the expressed antibodies, and screen antibodies with good sensitivity and specificity as the final selected monoclonal antibodies.

The pCMVp-NEO-BAN vector has a molecular weight of 6600 base pairs and is mainly composed of a CMVp promoter, a rabbit β-globulin gene intron, polyadenine, an ampicillin resistance gene, an anti-neo gene and a pBR322 backbone.

The antibody library was compared using the Ig BLAST system to determine the four framework regions (FR) and three complementarity determining regions (CDR) in the variable region of the antibody. The heavy chain and light chain of the antibody were submitted to the Kabat database respectively, and the Kabat number was used to represent the order of amino acids in the heavy chain CDR region and the light chain CDR region.

The amino acid sequences of the obtained heavy and light chains are as follows, wherein the CDR sequences are marked with bold and underline, the FR sequences are marked with italics, and the fragments without special marks are constant regions (see Table 3 for details).

table 3

The specific FR and CDR amino acid sequences are shown in Table 4 below:

Table 4

Example 3 Preparation and Binding Ability Test of CDR Region Single-point Mutation Antibodies

This example uses the pCMVp-NEO-BAN vector to construct the wild-type antifungal 1,3-β-D-glucan antibody full gene expression plasmid obtained in Example 2, and the restriction endonucleases used are AgeⅠ and HindⅢ. On this basis, the overlapping extension method is used to point-mutate the non-alanine amino acids in the CDR1, CDR2 and CDR3 regions of the wild-type antibody to alanine one by one. The successfully constructed heavy chain and light chain expression vectors are paired with the corresponding wild-type light chain and heavy chain expression vectors, respectively, and 293T cells are transfected. After 48 hours, the supernatant is collected by centrifugation to obtain antibodies with single amino acid mutations, and the antibodies are purified.

Antifungal 1,3-β-D-glucan monoclonal antibodies with different single point mutations and wild type were used as coating antibodies and enzyme-labeled antibodies to construct an ELISA detection system to detect fungal 1,3-β-D-glucan in samples. The steps are as follows:

(1) The antifungal 1,3-β-D-glucan monoclonal antibody was prepared into a coating solution with a concentration of 1000 ng/mL using 0.01 M carbonate buffer (CBS), and 100 μL/well was added to the microplate for overnight coating at 4°C. The next day, the coating solution was removed, the plate was blocked for 1 hour, and dried for 30 minutes to prepare an ELISA plate;

(2) preparing an HRP-labeled antifungal 1,3-β-D-glucan monoclonal antibody with a conjugate stabilizer to prepare an enzyme-labeled antibody at a concentration of 1000 ng/mL;

(3) Heat-treat the sample with EDTA-2Na, add 100 μL/well to the ELISA plate, incubate at 37°C for 20 min, wash, add 100 μL/well of the enzyme-labeled antibody to the ELISA plate, incubate at 37°C for 20 min, and wash;

(4) Add chromogenic substrate to the ELISA plate at 100 μL/well, incubate at 37°C for 10 min, and stop reading.

The binding ability of the wild-type WT antibody to the substrate was defined as the standard unit 1, and the binding ability of each mutant antibody was evaluated.

After the heavy chain was mutated at each point, the binding test results are shown in Figure 1. It can be seen from the figure that except for position 33 in VH-CDR1, the other positions were mutated to alanine, which significantly improved the binding of the antibody to 1,3-β-D-glucan. After positions 50, 51, 53, 55-57, 63 and 64 in VH-CDR2 were mutated to alanine, the binding of the antibody to 1,3-β-D-glucan was significantly improved. After positions 100, 101, 103, 108 and 109 in VH-CDR3 were mutated to alanine, the binding of the antibody to 1,3-β-D-glucan was significantly improved.

After the mutations at each point of the light chain, the binding test results are shown in Figure 2. It can be seen from the figure that except for positions 24, 30, 31, 33 and 34 in VL-CDR1, the mutations at other positions to alanine significantly improved the binding of the antibody to 1,3-β-D-glucan. Only the mutation at position 52 to alanine in VL-CDR2 significantly improved the binding of the antibody to 1,3-β-D-glucan. The mutations at positions 93, 95, 99, 100 and 103 in VL-CDR3 to alanine significantly improved the binding of the antibody to 1,3-β-D-glucan.

Example 4 Preparation of antibodies with multiple mutations in the CDR region

In this example, a homology search was performed using Protein BLAST of NCBI (National Center for Biotechnology Information) and it was determined that there were more than 30% homologous proteins in the database, so homology modeling was used.

The obtained wild-type WT antibody amino acid sequence was input into Swiss-model for homology modeling, and the obtained protein conformation was evaluated by 3D structure. The amino acids in the unallowed region in the pull-type diagram (as shown in Figure 3) accounted for 1.11%, and the SAVES Verify 3D scores were all greater than zero (as shown in Figures 4 and 5). The letter B before the site in the horizontal axis in Figure 4 represents one of the heavy chains (A or B), and the letter C before the site in the horizontal axis in Figure 5 represents one of the light chains (C or D). It can be seen from the scoring results that the protein conformation is reasonable and can be used for docking analysis.

Structural optimization and molecular docking were calculated using PyMOL and Discovery Studio to determine the docking pose with the lowest energy. Molecular dynamics simulations were calculated using Amber20. The binding energy between antigen and antibody was analyzed with reference to experimental data, and amino acids at specific active sites were selected to guide multiple mutations.

Based on the molecular docking and molecular dynamics simulation results shown in Figures 6 to 8, it can be seen from Figure 8 that the four residues VAL52 (heavy), PHE58 (heavy), PRO105 (heavy), and TYR106 (heavy) on the heavy chain mainly play a hydrophobic role. At the same time, the calculation of mutation energy also shows that the mutation energy of these amino acids varies greatly. The four residues TYR31 (light), ASN33 (light), ASN34 (light), and ARG55 (light) on the light chain are mainly connected to the antigen by hydrogen bonds. The change in mutation energy also shows that the hydrogen bond force is weaker than the hydrophobic effect. Based on this, the full gene sequence of the heavy chain and light chain of the double-point mutated antifungal 1,3-β-D-glucan monoclonal antibody was designed to construct an expression vector and co-transfected into 293T cells. After 48 hours of culture, the supernatant was harvested by centrifugation, and the antibody was purified to obtain an antibody with double-point amino acid mutations, with mutation sites of H108F and L93D.

The binding ability of the wild-type antibody obtained in Example 2 and the antibody with double mutation sites obtained in this example was detected by using the titer detection method in Example 1. The method is to dilute the wild-type antibody and the mutant antibody according to the molar concentration according to the dilution concentration in the table, add 100 μL/well to the ELISA plate, 37°C, and react for 1 hour; shake off the reaction solution, pat dry, wash 3 times with PBST, add PBS 1:6000 diluted goat anti-rabbit secondary antibody (Beijing Solebow Technology Co., Ltd., Product No.: SE134), 100 μL/well, 37°C, 45 min, shake off the reaction solution, pat dry, wash 5 times with PBST, add 100 μL TMB/well, 37°C, color development for 10 min, stop, read, and the results are shown in Table 5. It can be seen that the binding ability of the mutant antibody provided in this example is nearly 100 times higher than that of the wild-type antibody provided in Example 2.

table 5

Example 5 Chemiluminescent detection based on double-point mutation antifungal 1,3-β-D-glucan monoclonal antibody

In this example, the wild-type antifungal 1,3-β-D-glucan monoclonal antibody and the double-point mutant antifungal 1,3-β-D-glucan monoclonal antibody obtained in Example 4 were used as detection antibodies and signal antibodies to construct a chemiluminescence system to detect fungal 1,3-β-D-glucan in the sample. The steps are as follows:

(1) Add EDTA solution to the serum sample, heat at 120°C for 6 min, centrifuge at 10,000 g for 10 min, and obtain the supernatant as the test sample;

(2) adding the EDTA-pretreated test sample, carboxyl magnetic beads-coupled detection antibody, and acridine sulfonamide-labeled signal antibody to the reaction cup, mixing and incubating for 6 minutes to form an antibody-antigen sandwich immune complex;

(3) placing the mixed solution on a magnetic separator, removing the supernatant, and adding a washing solution to wash to remove substances not bound to the magnetic beads;

(4) After placing the reaction cup at the measuring point, add the excitation solution and mix evenly to perform chemiluminescence. Use a photomultiplier to detect the luminescence intensity. Analyze the concentration of fungal 1,3-β-D-glucan in the sample based on the chemiluminescence value and the pre-established standard curve (the sample is a gradient diluted positive standard).

In this example, the Limulus amebocyte lysate assay was used to detect the concentration of fungal 1,3-β-D-glucan in the same sample.

The results are shown in Table 6 and Figures 9 and 10. Samples with a detection concentration of ≥90 pg/mL are fungal 1,3-β-D-glucan-positive samples. It can be seen that the chemiluminescence detection method based on multi-point mutation antifungal 1,3-β-D-glucan monoclonal antibodies has a better correlation than the wild-type antibody and horseshoe crab reagent method (R ² >0.98).

Table 6

Example 6 Effect of sample processing method on test results

This embodiment uses different sample processing methods to pre-treat the sample, and uses the horseshoe crab reagent method and the chemiluminescence method to detect the concentration of fungal 1,3-β-D-glucan.

The results are shown in Tables 7 and 8. The concentration of fungal 1,3-β-D-glucan (pg/mL) can be detected by chemiluminescence method when the samples are treated with alkaline solution (1M KOH, 0.3M KCl) at 37°C, 0.12M EDTA-2Na at 100°C, or acidic sample release solution (10% HCl, 2% Gly) at 2-40°C. The detection results are well correlated with the Limulus amebocyte lysate method (R ² >0.98).

Table 7

Table 8

Example 7 Endotoxin Anti-interference Effect

In this example, different concentrations of endotoxin (Sigma, catalog number: 1235503) were added to the test sample, and carboxylated magnetic beads coupled antibodies were used as detection antibodies to detect the effect of endotoxin in the sample on the accuracy of the chemiluminescence system of Example 5.

The results are shown in Table 9 below. The detection time of the chemiluminescence method is significantly shorter than that of the horseshoe crab reagent method (50 minutes), requiring only 10 minutes. When the endotoxin concentration is added to the sample near the critical value to 640 ng/mL, the horseshoe crab reagent method detects a false positive, while the magnetic particle chemiluminescence method does not change the positive and negative test results of the sample. This shows that the use of carboxylated magnetic beads coupled to antibodies eliminates the interference of endotoxins in the sample on the results.

Table 9

Example 8 Immunofluorescence detection based on multi-point mutation antifungal 1,3-β-D-glucan monoclonal antibodies

This embodiment provides a 1,3-β-D-glucan detection test strip for detecting 1,3-β-D-glucan from different sources. The preparation method of the 1,3-β-D-glucan detection test strip includes:

(1) Processing sample pad:

The sample pad was treated with a sample pad treatment solution, the formula of which was 100 mmol/L Tris, 0.5 wt% PVP-K30, 0.5 wt% Tween-20 and 1 wt% BSA. The treated sample pad was dried at 37°C and 25% humidity for 4 hours.

(2) Preparation of fluorescent microsphere-labeled antibodies:

a. The fluorescent microsphere solution was mixed with 50 mmol / L MES buffer to obtain a fluorescent microsphere mixture with a fluorescent microsphere concentration of 0.1 wt%, and 10 mg / mL EDC solution and 10 mg / mL NHS solution were added to the fluorescent microsphere mixture, wherein the volume ratio of the fluorescent microsphere mixture, EDC solution and NHS solution was 100:1:1, and the fluorescent microspheres were activated by shaking at 25 ° C for 120 min, and the precipitate was collected by centrifugation at 10000g for 30 min to obtain activated fluorescent microspheres;

b. Dilute the activated fluorescent microspheres obtained in step a with 25 mmol/L HEPES solution, and then add avidin to mix, wherein the mass ratio of the activated fluorescent microspheres to the avidin is 20:1, and react at 25°C for 10 hours to obtain avidin-labeled fluorescent microspheres;

c. Treat the avidin-labeled fluorescent microspheres with a blocking solution at a concentration of 1 wt%, collect the precipitate by centrifugation, and obtain an avidin-fluorescent microsphere complex;

d. Biotin activated by N-hydroxysuccinimide ester was mixed with multi-point mutation antifungal 1,3-β-D-glucan monoclonal antibody at a molar ratio of 20:1, shaken at 25°C for 10 hours, and unbound biotin was removed to obtain a biotin-multi-point mutation antifungal 1,3-β-D-glucan monoclonal antibody complex; biotin activated by N-hydroxysuccinimide ester was mixed with chicken IgY antibody at a molar ratio of 20:1, shaken at 25°C for 10 hours, and unbound biotin was removed to obtain a biotin-chicken IgY antibody complex;

(3) Mixing the avidin-labeled fluorescent microspheres and the biotin-labeled antibodies and embedding them on a fluorescent pad:

The fluorescent microsphere-labeled avidin and the antibody-labeled biotin obtained in step (2) are mixed in a mass ratio of 10:1, the mass ratio of the avidin-fluorescent microsphere complex to the biotin-multi-point mutation antifungal 1,3-β-D-glucan monoclonal antibody complex is 10:1, and the mass ratio of the avidin-fluorescent microsphere complex to the biotin-chicken IgY antibody complex is 10:1. Spray them onto the fluorescent pad at a usage amount of 5 μL/cm and a spacing of 6 mm, and dry them at 37°C and a humidity of 25% for 2 h.

(4) Coating the test line and quality control line on the nitrocellulose membrane:

The multi-point mutation anti-fungal 1,3-β-D-glucan monoclonal antibody and goat anti-chicken IgY antibody were streaked on nitrocellulose membranes, respectively. The coating amount of bacterial 1,3-β-D-glucan monoclonal antibody was 1 μL/cm, and the coating amount of goat anti-chicken IgY antibody was 1 μL/cm, and they were dried at 37°C and 25% humidity for 10 h.

(5) Assembling the test strips:

A nitrocellulose membrane, a water absorbent pad, a fluorescent pad and a sample pad are sequentially pasted on a PVC bottom plate, wherein the water absorbent pad and the fluorescent pad each independently cover the nitrocellulose membrane by 2 mm, and the sample pad covers the fluorescent pad by 2 mm. After assembly, the test strips are cut to obtain the multi-point mutant antifungal 1,3-β-D-glucan detection test strips.

In this test example, 1,3-β-D-glucan test strips were used to detect polysaccharide antigens extracted from fungal strains from two different sources, including antigens extracted from Candida albicans (ATCC 90028) and Aspergillus fumigatus (ATCC 13073).

The statistical results of the sensitivity of the 1,3-β-D-glucan test strips to react with polysaccharide antigens extracted from fungal strains of two different sources are shown in Table 10.

Table 10

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Industrial Applicability

The antifungal 1,3-β-D-glucan binding protein provided by the present invention has high specificity and is applied in the chemiluminescence method, which greatly broadens its detection threshold, reduces its detection limit, and enhances its quantitative sensitivity. It not only has the advantage of high stability, but also is free from the interference of HAMA, and has excellent practicality in the field of in vitro diagnostic technology.

Claims (14)

1. A binding protein comprising a 1,3-β-D-glucan binding domain, characterized in that the antigen binding domain comprises at least one complementary determining region selected from the following amino acid sequences, or has at least 80% sequence identity with the complementary determining region of the following amino acid sequence and has a binding affinity with 1,3-β-D-glucan of KD≤10 ^-9 mol/L;

   The complementarity determining region CDR-VH1 is X1-X2-X3-W-X4-X5, wherein,

   X1 is N or A, X2 is D or A, X3 is F or A, X4 is I or A, and X5 is C or A;

   The complementary determining region CDR-VH2 is X1-X2-V-X3-D-X4-X5-X6-F-G-F-S-A-S-X7-A-K-G, wherein,

   X1 is C or A, X2 is M or A, X3 is P or A, X4 is G or A, X5 is S or A, X6 is G or A, X7 is W or A;

   The complementarity determining region CDR-VH3 is Y-X1-X2-V-X3-G-P-Y-S-X4-X5-X6, wherein

   X1 is G or A, X2 is D or A, X3 is G or A, X4 is F or A, X5 is K or A, X6 is I or A;

   The complementary determining region CDR-VL1 is Q-X1-X2-X3-X4-X5-G-Y-X6-N-N-X7-A, wherein

   X1 is S or A, X2 is S or A, X3 is Q or A, X4 is S or A, X5 is V or A, X6 is G or A, X7 is L or A;

   The complementary determining region CDR-VL2 is X1-A-S-R-L-A-S, wherein,

   X1 is G or A;

   The complementary determining region CDR-VL3 is A-G-X1-Y-X2-I-I-T-X3-X4-C-V-X5, wherein,

   X1 is D or A, X2 is G or A, X3 is D or A, X4 is T or A, and X5 is F or A.
2. The binding protein according to claim 1, characterized in that

   In the complementary determining region CDR-VH1, X3 is A;

   Alternatively, in the complementary determining region CDR-VH2, X3 is A;

   Alternatively, in the complementary determining region CDR-VH3, X4 is A;

   Alternatively, in the complementary determining region CDR-VL1, X2 is A;

   Alternatively, in the complementary determining region CDR-VL2, X1 is A;

   Alternatively, in the complementarity determining region CDR-VL3, X4 is A.
3. The binding protein according to claim 1 or 2, characterized in that the complementarity determining region comprises any combination of the following (a) to (i):

   (a) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL1, X1 is A;

   (b) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL2, X1 is A;

   (c) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL3, X1 is A;

   (d) in the complementary determining region CDR-VH3, X1 is A, and in the complementary determining region CDR-VL3, X4 is A;

   (e) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL1, X1 is A;

   (f) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL2, X1 is A;

   (g) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL3, X1 is A;

   (h) in the complementary determining region CDR-VH3, X4 is A, and in the complementary determining region CDR-VL3, X4 is A;

   (i) in the complementary determining region CDR-VH3, X1 is A and X4 is A; in the complementary determining region CDR-VL1, X1 is A; in the complementary determining region CDR-VL2, X1 is A; in the complementary determining region CDR-VL3, X1 is A and X4 is A.
4. The binding protein according to any one of claims 1 to 3, characterized in that the binding protein comprises at least 3 CDRs; or, the binding protein comprises 6 CDRs;

   Preferably, the binding protein is one of nanoantibodies, F(ab')2, Fab', Fab, Fv, scFv, bispecific antibodies and antibody minimum recognition units.
5. The binding protein according to any one of claims 1 to 3, characterized in that the binding protein comprises heavy chain backbone regions FR-L1, FR-L2, FR-L3 and FR-L4 whose sequences are sequentially shown in SEQ ID NOs: 1 to 4, and/or light chain backbone regions FR-H1, FR-H2, FR-H3 and FR-H4 whose sequences are sequentially shown in SEQ ID NOs: 5 to 8;

   Preferably, the binding protein further comprises an antibody constant region sequence;

   Preferably, the constant region sequence is selected from any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD;

   Preferably, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human;

   Preferably, the constant region is derived from rabbit.
6. The biomaterial is characterized in that the biomaterial comprises any one of the following:

   (a) a nucleic acid molecule encoding the binding protein according to any one of claims 1 to 5;

   (b) a vector comprising the nucleic acid molecule described in (a);

   (c) a host cell comprising the nucleic acid molecule of (a) or the vector of (b).
7. The method for preparing the binding protein according to any one of claims 1 to 5 is characterized in that the preparation method comprises culturing the host cell according to claim 6, and recovering the produced binding protein from the culture medium or from the cultured host cell.
8. Use of the binding protein according to any one of claims 1 to 5 in the preparation of a diagnostic agent, a test strip or a kit for diagnosing fungal infection.
9. A 1,3-β-D-glucan detection test strip, characterized in that the detection line of the test strip is obtained by streaking the binding protein according to any one of claims 1 to 5.
10. A reagent or a kit for detecting fungal 1,3-β-D-glucan, characterized in that the reagent or the kit comprises the binding protein according to any one of claims 1 to 5.
11. The binding protein according to any one of claims 1 to 5, or the 1,3-β-D-glucan detection test strip according to claim 9, or the reagent or kit for detecting fungal 1,3-β-D-glucan according to claim 10, for use in diagnosing fungal infection.
12. A method for detecting 1,3-β-D-glucan for non-diagnostic purposes, characterized in that the detection method comprises chemiluminescence detection, the chemiluminescence detection comprises a pretreatment step of the sample to be tested, and the pretreatment step comprises any one of (a) to (c):

    (a) Treat the sample with alkali solution at 37°C;

    (b) The sample was treated with EDTA-2Na solution at 100°C;

    (c) treating the sample to be tested with the acidic sample release solution at 2 to 40° C.;

    Alternatively, the detection method comprises detection using the test strip of claim 9;

    The 1,3-β-D-glucan source includes Candida albicans or Aspergillus fumigatus.
13. A method for detecting fungi, comprising:

    A) contacting the binding protein of any one of 1 to 5, or the 1,3-β-D-glucan detection test strip of claim 9, or the reagent or kit for detecting fungal 1,3-β-D-glucan of claim 10 with a sample from the subject under conditions sufficient for a binding reaction to occur, so as to perform a binding reaction; and

    B) Detection of immune complexes produced by the binding reaction.
14. A method for diagnosing a subject with a fungal infection or a disease associated with a fungal infection, comprising:

    A) contacting the binding protein of any one of 1 to 5, or the 1,3-β-D-glucan detection test strip of claim 9, or the reagent or kit for detecting fungal 1,3-β-D-glucan of claim 10 with a sample from the subject under conditions sufficient for a binding reaction to occur, so as to perform a binding reaction; and

    B) Detection of immune complexes produced by the binding reaction.