CN116041499A

CN116041499A - 1, 3-beta-D-glucan binding protein, preparation method and application

Info

Publication number: CN116041499A
Application number: CN202211737487.2A
Authority: CN
Inventors: 刘春龙; 付成华; 刘昶; 粟艳; 周泽奇
Original assignee: Dana Tianjin Medical Laboratory Co ltd; Dana Hunan Biotechnology Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dana Tianjin Medical Laboratory Co ltd; Dana Hunan Biotechnology Co ltd; Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-05-02

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to 1, 3-beta-D-glucan binding protein, a preparation method and application. The antifungal 1, 3-beta-D-glucan binding protein provided by the invention has high specificity, the monoclonal antibody is a gene expression product, after 3 Complementarity Determining Regions (CDR) of a wild type binding protein are determined, all non-alanine amino acids in the CDR region are further subjected to point mutation into alanine one by one, and the binding force of different mutants and a substrate is compared, so that the antibody with the binding force of the substrate being 100 times that of the wild type binding protein is obtained on the basis of the wild type binding protein. The method is applied to a chemiluminescence method, so 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 simultaneously avoids the influence of HAMA interference.

Description

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

Technical Field

The invention relates to the technical field of in-vitro diagnosis, in particular to 1, 3-beta-D-glucan binding protein, a preparation method and application.

Background

The current fungus 1, 3-beta-D-glucan detection technology is limulus reagent method detection, and the detection time is about 50 minutes. The key raw material of the limulus reagent is from a national secondary wild protection animal: the limulus reagent is extracted from limulus blood. And the growth cycle of the horseshoe crab is long, the horseshoe crab is extremely difficult to breed, the horseshoe crab can only be used for collecting blood by fishing the wild horseshoe crab, and each horseshoe crab can be used for taking 100-300 mL of blood. After years of killing, horseshoe crab has become an endangered species, and the raw material of horseshoe crab reagent is at risk of under-supply. In addition, the difference between the limulus reagent batches prepared from natural limulus blood is large, so that the production cost of the product is increased and the reproducibility is poor. The detection principle of the limulus reagent method is as follows: the 1, 3-beta-D-glucan can specifically activate the factor G in the limulus reagent, so as to activate the clotting zymogen to form clotting enzyme, and the clotting enzyme catalyzes subsequent color reaction or turbidity reaction. Taking fungus 1, 3-beta-D-glucan detection product of Dana (Tianjin) biotechnology Co., ltd as an example, the kit adopts a limulus reagent chromogenic method, firstly, a sample is pretreated for 10min, and then limulus reagent and chromogenic substrate are added for incubation for 40min. The 1, 3-beta-D-glucan can specifically activate G factor in a reaction main agent, further activate prothrombin, and coagulate chromogenic substrate in hydrolysis reaction of enzyme to generate free paranitroaniline (pNA) so as to cause absorbance change, and the concentration of the 1, 3-beta-D-glucan is quantified according to the dynamic detection of the absorbance change rate of the solution. The total time of the method was about 50min, with a linear lower limit of 37.5pg/mL.

However, the limulus reagent also activates clotting zymogens to form clotting enzymes under the action of bacterial endotoxins, which are widely present in nature, and thus are highly susceptible to interference by bacterial endotoxins.

In the method for detecting 1, 3-beta-D-glucan by using the limulus reagent instead, the binding force between the antibody and the substrate is limited, and the natural attribute is mostly reserved without modifying the system. Taking PCT detection product of Roche company as an example, the reagent adopts a sandwich method, a sample, a biotin-labeled antibody and a ruthenium complex-labeled antibody are added into a reaction cup, and an antigen-antibody sandwich complex is formed after incubation for a period of time. And adding streptavidin-coated magnetic bead particles for incubation, wherein the complex is combined with the magnetic beads through the action of biotin and streptomycin. The reaction liquid is sucked into the measuring pool, and the magnetic beads are adsorbed on the surface of the electrode through electromagnetic action. The substances which are not combined with the magnetic beads are removed through the ProCell, a certain voltage is applied to the electrode, the complex is subjected to chemiluminescence, the luminous intensity is measured through the photomultiplier, the luminous value is positively correlated with the concentration of the to-be-detected substance in the sample, the total time of the method is about 18min, the detection limit is low, the method is mainly limited by the binding capacity of the antibody and the substrate, and the sensitivity and the specificity of the antibody in clinical application are directly influenced. Therefore, there is a need for more excellent detection reagents and detection methods.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a binding protein of antifungal 1, 3-beta-D-glucan with high specificity, which is expected to have high binding force with the 1, 3-beta-D-glucan, can be applied to detection by a chemiluminescence method, a test strip method and the like, and is expected to greatly widen a detection threshold, reduce a detection lower limit, enhance quantitative sensitivity and avoid the influence of HAMA interference.

In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:

in a first aspect, the invention provides a binding protein comprising a 1,3- β -D-glucan binding domain comprising at least one complementarity determining region selected from the group consisting of, or having at least 80% sequence identity to, the complementarity determining region of the amino acid sequence and having KD.ltoreq.10 with 1,3- β -D-glucan ^-9 Binding force of 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 complementarity 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, and 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, and X6 is I or A;

the complementarity 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, and X7 is L or A;

the complementarity determining region CDR-VL2 is X1-A-S-R-L-A-S, wherein,

x1 is G or A;

the complementarity 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 alternative embodiment, in said complementarity determining region CDR-VH1, X3 is a;

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

alternatively, in the complementarity determining region CDR-VH2, X3 is A;

alternatively, in the complementarity determining region CDR-VH3, X4 is A;

alternatively, in the complementarity determining region CDR-VL1, X2 is A;

alternatively, in the complementarity determining region CDR-VL2, X1 is A;

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

In an alternative embodiment, the complementarity determining regions include any combination of (a) to (i) below:

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

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

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

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

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

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

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

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

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

In alternative embodiments, the binding protein comprises at least 3 CDRs therein; alternatively, the binding protein comprises 6 CDRs.

It is well known in the art that both binding specificity and avidity of antibodies are determined primarily by CDR sequences, and that amino acid sequences of non-CDR regions can be readily altered to obtain variants with similar biological activity according to well-established and well-known techniques. Thus, the invention also includes "functional derivatives" of the binding proteins. "functional derivative" refers to a variant obtained by deletion, substitution or insertion of one or more amino acid residues, one functional derivative retaining the activity of an antibody capable of binding 1, 3-beta-D-glucan. "functional derivatives" may comprise "variants" and "fragments" which possess similar biological activity as they possess the exact same CDR sequences as the binding proteins described herein.

In some embodiments, 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 to a complementarity determining region of an amino acid sequence and has a KD of less than or equal to 10 to 1, 3-beta-D-glucan ^-9 The bonding force of mol/L and KD value can be 10 ^- ⁸ mol/L、10 ^-7 mol/L, etc.

Preferably, the binding protein is one of nanobody, F (ab ') 2, fab', fab, fv, scFv, bispecific antibody and antibody minimal recognition unit.

In alternative embodiments, the binding protein comprises heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4, which are shown in sequence SEQ ID NO. 1-4, and/or light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4, which are shown in sequence SEQ ID NO. 5-8.

FR-H1	SEQ ID NO:1	QSLQESGGGLVQPGASLTLTCKASEFSFN
			FR-H2	SEQ ID NO:2	WVRQAPGKGLEWIA
FR-H3	SEQ ID NO:3	RFTISRTSSTTMTLQMTSLTAADTATYFCTR
			FR-H4	SEQ ID NO:4	WGPGTLVTVSS
FR-L1	SEQ ID NO:5	AAVLTQTPSPVSAAVGGTVTISC
			FR-L2	SEQ ID NO:6	WYQQKPGQPPKLLIY
FR-L3	SEQ ID NO:7	GVPSRFSGSGSGTQFTLTINGVQCDDAATYYC
			FR-L4	SEQ ID NO:8	FGGGTEVVVK

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

Preferably, the constant region sequence is selected from the group consisting of the sequence of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, igD constant regions.

Preferably, the constant region is of a species origin of cow, 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.

Preferably, the constant region of the heavy chain has an amino acid sequence of GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 9).

Preferably, the constant region of the light chain has an amino acid sequence of GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQ NSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 10).

In a second aspect, the present invention provides a biomaterial comprising any one of the following:

(a) A nucleic acid molecule encoding a binding protein according to any one of the embodiments provided in the first aspect;

(b) A vector comprising the nucleic acid molecule of (a);

(c) A host cell comprising (a) said nucleic acid molecule or (b) said vector.

The nucleic acid molecules comprise conservatively substituted variants thereof (e.g., the substitution 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, e.g., oligonucleotides.

In the vector, the nucleic acid sequence therein is operably linked to at least one regulatory sequence. "operably linked" refers to a coding sequence that is linked to regulatory sequences in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct expression of the protein of interest in a suitable host cell, and include promoters, enhancers and other expression control elements.

The vector may refer to a molecule or agent comprising a nucleic acid molecule of the invention or a fragment thereof, which is capable of carrying genetic information and which may deliver the genetic information into a cell. Typical vectors include plasmids, viruses, phages, cosmids, and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, the cell may be propagated and the cell may be selected for the presence or absence of the genetic information) or an expression vector (i.e., a vector comprising the necessary genetic elements to allow expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains regulatory elements necessary to effect expression in the specified target cell.

The nucleic acid molecules of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragments of the invention, such novel vectors also being part of the invention. The vector may include a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning vectors and expression vectors of the present invention are capable of spontaneous replication and thus can provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may include a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a signal peptide that causes secretion or integration of the peptide expression product onto a membrane, the nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a producer strain or cell line, the vector may or may not be integrated into the host cell genome when introduced into the host cell. The vector typically carries a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

The expression vectors of the invention are useful for transforming host cells. Such transformed cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the polypeptides of the invention. Transformed cells of the invention include microorganisms such as bacteria (e.g., E.coli, bacillus, etc.). Host cells also include cells derived from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably mammalian derived cells, e.g., CHO cells. The transformed cell is capable of replicating the nucleic acid fragment of the invention. When the peptide combinations of the invention are recombinantly produced, the expression product may be exported into culture medium or carried on the surface of the transformed cells.

In a third aspect, the present invention provides a method of producing a binding protein according to any one of the embodiments provided in the first aspect, the method comprising culturing a host cell according to the second aspect, recovering the produced binding protein from the culture medium or from the cultured host cell.

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

Construction of a suitable vector containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailing and religated as desired. Mutations may be introduced into the coding sequence by any method to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions, etc.

In a fourth aspect, the present invention provides the use of a binding protein according to any one of the embodiments of the first aspect for the preparation of a diagnostic agent, test strip or kit for diagnosing a fungal infection.

In a fifth aspect, the invention provides a 1,3- β -D-glucan detection test strip whose detection line is scored by a binding protein according to any one of the embodiments provided in the first aspect.

In a sixth aspect, the present invention provides a method for detecting 1,3- β -D-glucan for non-diagnostic purposes, the method comprising a chemiluminescent assay comprising a pretreatment step of a sample to be tested, the pretreatment step comprising any one of (a) to (c):

(a) Treating a sample to be tested by alkali liquor at 37 ℃;

(b) EDTA-2Na solution is used for treating a sample to be tested at 100 ℃;

(c) Treating a sample to be tested by the acidic sample release liquid at the temperature of 2-40 ℃;

alternatively, the detection method comprises detecting using the test strip of the fifth aspect;

the source of 1, 3-beta-D-glucan comprises Candida albicans or Aspergillus fumigatus.

In addition, the invention also provides a method for detecting 1, 3-beta-D-glucan in a sample to be detected, which comprises the following steps:

(a) Contacting 1,3- β -D-glucan in the test sample with a binding protein of any one of the first aspect-providing embodiments to form an immune complex; and

(b) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the 1,3- β -D-glucan in the test sample;

in this embodiment, the binding protein may be labeled with an indicator that shows signal strength to allow the complex to be readily detected.

In an alternative embodiment, in step (a), a second antibody is further included in the immunocomplex, the second antibody binding to the binding protein;

in alternative embodiments, the binding protein forms a partner antibody in the form of a first antibody with the second antibody for binding to a different epitope of 1,3- β -D-glucan;

the secondary antibody may be labeled with an indicator that shows signal strength to allow the complex to be readily detected.

In an alternative embodiment, in step (a), a second antibody is further included in the immunocomplex, the second antibody binding to the 1,3- β -D-glucan;

in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator that shows signal strength, so that the complex is easily detected.

In alternative embodiments, the indicator that displays signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.

In an alternative embodiment, the fluorescent substance comprises Alexa 350, alexa 405, alexa 430, alexa488, 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' -dimethoxy fluorescein, 5-carboxy-2 ',4',5',7' -Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethyl rhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol purple, light cresol Blue, para-aminobenzoic acid, erythrosin, phthalocyanine, cyanine, alternet-2-oxa-3-diazole azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyl diamine europium, europium cryptate or chelate, diamine, bis anthocyanin, la Jolla Blue dye, allophycocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine or texas red.

In an alternative embodiment, the radioisotope includes 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.

In alternative embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

In an alternative embodiment, the fluorescent microsphere is: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.

The antifungal 1, 3-beta-D-glucan binding protein provided by the invention has high specificity, the monoclonal antibody is a gene expression product, after 3 Complementarity Determining Regions (CDR) of a wild type binding protein are determined, all non-alanine amino acids in the CDR region are further subjected to point mutation into alanine one by one, and the binding force of different mutants and a substrate is compared, so that the antibody with the binding force of the substrate being 100 times that of the wild type binding protein is obtained on the basis of the wild type binding protein. The method is applied to a chemiluminescence method, so 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 advantages of high stability, small batch-to-batch difference and no influence of cell strain degeneration, can be expressed by selecting different animal-derived vectors, greatly improves the antibody production yield, reduces the production cost and avoids the influence of HAMA interference.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the effect of mutations at various points on the heavy chain on binding force in example 3 of the present invention;

FIG. 2 is the result of the effect of mutations at various points on the light chain on binding force in example 3 of the present invention;

FIG. 3 is a 3D structural drawing of a wild-type WT antibody of example 4 of the present invention;

FIG. 4 is a heavy chain SAVES version 3D scoring result for wild-type WT antibodies of example 4 of the present invention;

FIG. 5 is a light chain SAVES version 3D scoring result for wild-type WT antibodies of example 4 of the present invention;

FIG. 6 is a graph showing the comparison of the results of the heavy chain docking and the experimental results in example 4 of the present invention;

FIG. 7 is a graph showing the comparison of the results of the light chain docking and the experimental results in example 4 of the present invention;

FIG. 8 is a schematic representation of the binding of wild-type antibodies and antigens obtained in example 4 of the present invention;

FIG. 9 is a linear result of wild-type antibody detection obtained in example 5 of the present invention;

FIG. 10 shows the linear results of mutant antibody detection obtained in example 5 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

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

The term "amino acid" refers to a naturally occurring or non-naturally occurring carboxy alpha-amino acid. The term "amino acid" as used herein may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: A1a, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, c), glutamine (G1N, Q), glutamic acid (G1 u, 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 (Va 1, V). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocysteine, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

The term "binding protein comprising a 1,3- β -D-glucan binding domain" broadly refers to a protein or protein fragment capable of specifically binding to 1,3- β -D-glucan and comprising a CDR region.

The term "antibody", or immunoglobulin, includes polyclonal antibodies, monoclonal antibodies, and antigen compound binding fragments of such antibodies, including Fab, F (ab') 2, fd, fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as single chain derivatives of such antibodies and fragments. The type of antibody may be selected from IgG1, igG2, igG3, igG4, igA, igM, igE or IgD. Furthermore, "antibodies" include naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms).

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

As used herein, a "framework" or "FR" region refers to the contiguous regions (FR 1, FR2, FR3, and FR 4) of each antibody variable domain framework separated by CDRs into which it is further subdivided.

Typically, the variable regions VL/VH of the heavy and light chains are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.

EXAMPLE 1 fungal 1, 3-beta-D-glucan immunization test Rabbit

Subcutaneous immunization of experimental rabbits (50. Mu.g/dose) with fungal 1, 3-beta-D-glucan conjugated with BSA or KLH (Shanghai Prime Biotech Co., ltd., cat# E-BGOSAG), the first immunization with fungal 1, 3-beta-D-glucan emulsified with complete Freund's adjuvant, followed by the second, third, fourth, fifth, sixth and seventh immunization with fungal 1, 3-beta-D-glucan emulsified with incomplete Freund's adjuvant every other week; seven times of immunity are followed by booster immunization by pure antigen auricular intravenous injection, rabbit blood is taken, ELISA is used for measuring serum titer of gradient dilution, and rabbits with specific antibody titer exceeding 500000 in serum are screened. The ELISA assay comprises the following specific steps: 1, 3-beta-D-glucan 0.2 mug/ml, 100 mug/hole, 80 ℃ coating ELISA plate, 5% skim milk powder, 200 mug/hole, 37 ℃ sealing for 2 hours, throwing away sealing liquid, and 37 ℃ drying for standby. The rabbits were collected with blood at 3000 rpm, serum was collected after centrifugation from 1:10000 started to be diluted to 1 with PBS: 1280000, respectively adding an ELISA plate, 100 mu L/hole, and reacting for 1h at 37 ℃; throwing away the reaction liquid, beating to dry, washing with PBST for 3 times, adding PBS 1: 6000-fold diluted goat anti-rabbit secondary antibody (Beijing Soy Bao technology Co., ltd., product number: SE 134), 100. Mu.L/well, 37 ℃,45min, throwing away the reaction solution, beating to dry, PBST washing 5 times, adding 100. Mu.L TMB/well, 37 ℃, developing for 10min, stopping, reading, and rabbit serum titer detection results are shown in the following table:

Example 2 preparation of anti-fungal 1, 3-beta-D-glucan antibodies

Based on example 1, meltRabbits with an immune titer exceeding 500000 were boosted three days before the combination and vaccinated in the same amount as the previous immunization without adjuvant and by auricular intravenous injection. Feeder cells were prepared the day before fusion, 10mL of HAT selection medium was aspirated with a sterile syringe and injected into the rabbit abdominal cavity, the abdomen was gently rubbed with an alcohol cotton ball, and the medium was withdrawn. Adding into 40mL HAT culture solution, spreading into 4 96-well cell culture plates, 100 μl/well, 37deg.C, 5% CO ₂ Culturing in a cell culture incubator. One week prior to fusion, myeloma cells (Sp 2/0 cells) were resuscitated and cultured in PRMI-1640 medium containing 10% fetal bovine serum, 37℃and 5% CO ₂ Subculturing in an incubator. Collecting cells in logarithmic phase into centrifuge tube, counting, diluting to 10 ⁷ The sample is ready for use. The rabbits immunized for 3 days were taken, the spleens were aseptically removed on a clean bench, washed several times in sterile dishes, and connective tissue was stripped. Placing spleen on a microporous copper mesh, adding fresh RPMI-1640 culture solution, sucking the culture solution by a syringe, injecting the culture solution from the spleen for one time, blowing spleen cells, repeating for several times, and lightly grinding the rest spleen by using an inner plug of the syringe until no obvious red tissue block exists. The spleen cell suspension in the plate is gently blown and transferred into a 50mL centrifuge tube, centrifuged at 1000r/min for 5min, and the spleen cells are collected and counted for later use. The spleen cells of the immunized rabbits were combined with Sp2/0 cells in a cell number of 10:1, adding into a 50mL centrifuge tube, centrifuging at 1000r/min for 5min, discarding the supernatant, lightly rubbing at the palm to fully mix the two cells, placing the centrifuge tube into a 100mL blue-capped bottle, filling hot water at 37 ℃ into the blue-capped bottle, dropwise adding preheated 1mL DMSO/PEG into a fusion tube within 1min, slowly and quickly, and lightly rotating the centrifuge tube while adding. Then, the reaction was terminated by immediately adding the antibiotic-free and bloodless RPMI-1640 medium, 1mL for the first minute, zhong Jia mL for the second minute, 3mL for the third minute, and 4mL for the fourth minute. Centrifuging at 37deg.C in water bath for 5min at 800r/min for 5min, removing supernatant, suspending the precipitate with HAT, mixing to 40mL HAT selective culture solution containing pre-heated 20% calf serum at 37deg.C, spreading into 96-well cell plate with feeder cells at 100 μl/well, placing the culture plate into 37 deg.C, 5% CO ₂ Culturing in an incubator. After 7 days, the cell plates were half-changed with fresh HAT medium and 10 days laterHT medium was completely changed. Cells positive for detection in 96-well plates were subcloned by limiting dilution: firstly preparing feeder cells according to the method, taking hybridoma cells to be cloned for cell count, diluting the cells to 5-8 cells/ml by using HT culture medium, adding 100 mu L/hole into 96-well cell plates paved with feeder cells, cloning one 96-well cell plate for each hybridoma cell, and controlling the temperature to 37 ℃ and the concentration of CO to 5% ₂ Culturing in a cell culture incubator. After about 5 days, the number of clones in the cell well is counted, marked, 7 days later and a new culture medium is changed, and the cell is detected when 1/3-1/2 of the whole bottom of the well is paved. After cloning for 2-3 times, when all the cell holes of the 96-well plate are positive, the amplified culture, strain fixing and freezing storage can be carried out. The hybridoma cells of the positive determination fixed strain are subjected to amplification culture and frozen, and the specific process is as follows: the well-grown hybridoma cells were gently blown down from the cell flask with the use of the anti-bloodless DMEM, centrifuged at 1000r/min for 5min, and the supernatant was discarded. Adding the frozen stock solution (containing 40% RPMI-1640 culture solution, 50% fetal calf serum and 10% DMSO), blowing off the cells, and packaging into cell frozen stock tubes. The frozen tube was placed in a frozen box in a-70 ℃ refrigerator and transferred into liquid nitrogen one day later. After cloning genes of specific antibodies from the cell strains, constructing a recombinant expression vector pCMVp-NEO-BAN-Ab containing the genes of the specific antibodies, transforming the recombinant expression vector into CHO cells for expression, purifying the expressed antibodies, and screening the antibodies with good sensitivity and specificity as the finally selected monoclonal antibodies.

The pCMVp-NEO-BAN vector: the molecular weight is 6600 base pairs, and mainly comprises CMVp promoter, rabbit beta-globulin gene intron, poly adenine, ampicillin resistance gene, neo resistance gene and pBR322 skeleton.

The antibody library was compared using the Ig BLAST system to determine 4 Framework Regions (FRs) and 3 Complementarity Determining Regions (CDRs) in the variable region of the antibody. The heavy chain and the light chain of the antibody are submitted to a Kabat database, and the arrangement sequence of each amino acid in the heavy chain CDR region and the light chain CDR region is represented by Kabat numbering.

The amino acid sequences of the heavy and light chains obtained are as follows, wherein the CDR sequences are indicated by bold and underlined, the FR sequences are indicated in italics, and the fragments which are not specifically marked are constant regions.

Specific FR and CDR amino acid sequences are as follows:

example 3 preparation of CDR region Single point mutant antibodies and binding force test

In this example, the wild-type antifungal 1, 3-. Beta. -D-glucan antibody whole-gene expression plasmid obtained in example 2 was constructed using pCMVp-NEO-BAN vector, and restriction enzymes were used as both AgeI and HindIII. Based on this, the amino acids other than alanine in the CDR1 region, CDR2 region and CDR3 region of the wild-type antibody were point-mutated to alanine by the overlap extension method. And pairing the heavy chain and light chain expression vectors which are successfully constructed with corresponding wild-type light chain and heavy chain expression vectors respectively, transfecting 293T cells, centrifuging the supernatant after 48 hours to obtain antibodies with single amino acid mutation, and purifying the antibodies.

Different single-point mutation and wild antifungal 1, 3-beta-D-glucan monoclonal antibodies are used as coating antibodies and enzyme-labeled antibodies, an ELISA detection system is constructed, and fungus 1, 3-beta-D-glucan in a sample is detected, and the steps are as follows:

(1) Preparing an antifungal 1, 3-beta-D-glucan monoclonal antibody into coating liquid with the concentration of 1000ng/mL by using 0.01M Carbonate Buffer Solution (CBS), adding 100 mu L/hole into a micro-pore plate, coating overnight at 4 ℃, removing the coating liquid the next day, sealing for 1h, and drying for 30min to obtain the ELISA plate;

(2) Preparing an enzyme-labeled antibody with the concentration of 1000ng/mL by using a conjugate stabilizer for the HRP-labeled antifungal 1, 3-beta-D-glucan monoclonal antibody;

(3) Heat-treating a sample with EDTA-2Na, adding 100 mu L/hole into an ELISA plate, incubating at 37 ℃ for 20min, washing, adding an ELISA antibody into the ELISA plate at 100 mu L/hole, incubating at 37 ℃ for 20min, and washing;

(4) Chromogenic substrate was added to the ELISA plate at 100. Mu.L/well and incubated at 37℃for 10min, and the reading was terminated.

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

After each point of the heavy chain is mutated, the binding force detection result is shown in figure 1, and the binding force of the antibody and 1, 3-beta-D-glucan is obviously improved after each point of the VH-CDR1 is mutated into alanine except for 33 positions. After 50 th, 51 st, 53 rd, 55-57 th, 63 rd and 64 th of VH-CDR2 are mutated into alanine, the binding force of the antibody and 1, 3-beta-D-glucan is obviously improved. After the 100 th, 101 th, 103 th, 108 th and 109 th of VH-CDR3 are mutated into alanine, the binding force of the antibody and 1, 3-beta-D-glucan is obviously improved.

After each point of the light chain is mutated, the binding force detection result is shown in figure 2, and the figure shows that after each point of the VL-CDR1 is mutated into alanine except for 24, 30, 31, 33 and 34, the binding force of the antibody and 1, 3-beta-D-glucan is obviously improved. After only 52 th position in VL-CDR2 is mutated into alanine, the binding force between the antibody and 1, 3-beta-D-glucan can be obviously improved. After the 93 rd, 95 th, 99 th, 100 th and 103 th mutation in VL-CDR3 is changed into alanine, the binding force of the antibody and 1, 3-beta-D-glucan is obviously improved.

EXAMPLE 4 preparation of CDR region Multipoint mutant antibodies

In this example, homology search was performed by Protein BLAST from NCBI (National Center for Biotechnology Information ), and it was determined that more than 30% of homologous proteins exist in the database, so homology modeling was selected.

The obtained wild-type WT antibody amino acid sequence was input into Swiss-model for homology modeling, and the obtained protein conformation was subjected to 3D structure evaluation. The ratio of the amino acids in the disallowed region in the pull graph (shown in fig. 3) is 1.11%, the SAVES version 3D score is greater than zero (shown in fig. 4 and 5), the letter B before the position in the abscissa in fig. 4 represents one of the heavy chains (A or B), the letter C before the position in the abscissa in fig. 5 represents one of the light chains (C or D), and the protein conformation is reasonable as seen from the scoring result and can be used for docking analysis.

The structure optimization and molecular docking are calculated by using PyMOL and Discovery Studio, the lowest energy docking post is determined, the molecular dynamics simulation is calculated by using Amber20, the binding energy between antigen and antibody is analyzed by taking experimental data as a reference, and the amino acid of a specific active site is selected to guide multiple mutation.

Based on the results of molecular docking and molecular dynamics simulation, as shown in fig. 6-8, it can be seen from fig. 8 that four residues of VAL52 (heavy), PHE58 (heavy), PRO105 (heavy) and TYR106 (heavy) on the heavy chain mainly play a hydrophobic role, and meanwhile, the mutation energy of the several amino acids is greatly changed in calculation of mutation energy. The four residues of TYR31 (light), ASN33 (light), ASN34 (light) and ARG55 (light) on the light chain are connected with antigen mainly by hydrogen bonds, and the hydrogen bond acting force is weaker than the hydrophobic effect through the change of mutation energy. Based on the sequence, the whole gene sequences of the heavy chain and the light chain of the antifungal 1, 3-beta-D-glucan monoclonal antibody with double-point mutation are designed and used for constructing an expression vector, 293T cells are transfected together, supernatant is obtained after 48H of culture, and the antibody is purified to obtain the antibody with double-point amino acid mutation, wherein mutation sites are H108F and L93D.

The binding force of the wild-type antibody obtained in example 2 and the antibody of the double mutation site obtained in this example was detected by the method of titer detection in example 1, specifically by diluting the wild-type antibody and the mutant antibody according to the molar concentration in the table, adding an ELISA plate, and reacting at 37℃for 1h; throwing away the reaction liquid, beating to dry, washing with PBST for 3 times, adding PBS 1: 6000-fold dilution of goat anti-rabbit secondary antibody (Beijing Soy Bao technology Co., ltd., product No.: SE 134), 100. Mu.L/well, 37 ℃,45min, throwing away the reaction solution, taking a pat dry, PBST washing 5 times, adding 100. Mu.L TMB/well, 37 ℃, developing for 10min, stopping, reading, and the result shows that the binding force of the mutant antibody provided in this example is improved by nearly 100-fold compared with that of the wild-type antibody provided in example 2.

Dilution (mol/L)	Wild type antibody	Mutant antibodies
			1.00E-03	3.873	3.969
1.00E-04	3.784	3.919
			1.00E-05	3.558	3.779
1.00E-06	3.056	3.687
			1.00E-07	2.513	3.265
1.00E-08	1.629	3.026
			1.00E-09	0.825	2.462
1.00E-10	0.415	1.532
			1.00E-11	0.213	0.865

Example 5 chemiluminescent detection of anti-fungal 1, 3-beta-D-glucan monoclonal antibody based on double point mutations

In the embodiment, a chemiluminescent system is constructed by using a wild type antifungal 1, 3-beta-D-glucan monoclonal antibody and the double point mutation antifungal 1, 3-beta-D-glucan monoclonal antibody obtained in the embodiment 4 as detection antibodies and signal antibodies, and the detection of the fungus 1, 3-beta-D-glucan in a sample is carried out, wherein the steps are as follows:

(1) Adding EDTA solution into the serum sample, heating at 120 ℃ for 6min, centrifuging 10000g for 10min, and obtaining a supernatant as a detection sample;

(2) Adding an EDTA pretreated detection sample, a carboxyl magnetic bead coupled detection antibody and an acridine sulfonamide labeled signal antibody into a reaction cup, mixing and incubating for 6min to form an antibody-antigen sandwich immune complex;

(3) Placing the mixed solution on a magnetic separator, sucking the supernatant, and adding a washing solution for washing to remove substances which are not combined with the magnetic beads;

(4) After the reaction cup is placed at a measuring point, adding an excitation solution, uniformly mixing, carrying out chemiluminescence, detecting the luminous intensity by using a photomultiplier, and analyzing the concentration of the fungus 1, 3-beta-D-glucan in the sample according to the chemiluminescence numerical value and a standard curve (the sample is a positive standard product of gradient dilution) established in advance.

The present example simultaneously uses the limulus reagent method to detect the concentration of fungal 1, 3-beta-D-glucan in the same sample.

As a result, the results are shown in the following Table, and as shown in FIG. 9 and FIG. 10, the sample with a detection concentration of 90pg/mL or more is a fungus 1, 3-beta-D-glucan positive sample, and it can be seen that the chemiluminescence detection method based on the multipoint mutation antifungal 1, 3-beta-D-glucan monoclonal antibody is better correlated with the detection results of the wild-type antibody and the limulus reagent method (R) ² >0.98)。

Example 6 Effect of sample processing method on detection results

In this example, samples were pretreated by different sample treatment methods, and the concentration of fungal 1,3- β -D-glucan was measured by limulus reagent method and chemiluminescent method, respectively.

As shown in the following Table, the concentration (pg/mL) of 1, 3-beta-D-glucan in fungi can be detected by chemiluminescent method by treating the sample with alkaline solution (1M KOH,0.3M KCl) at 37℃and 0.12M EDTA-2Na at 100℃or acidic sample releasing solution (10% HCl,2% Gly) at 2-40℃and the detection result has good correlation with the limulus reagent method (R ² >0.98)

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EXAMPLE 7 endotoxin anti-interference Effect

In this example, endotoxin (Sigma, cat# 1235503) was added to the test sample at various concentrations, and the effect of endotoxin in the sample on the accuracy of the chemiluminescent system of example 5 was detected using carboxylated magnetic bead conjugated antibodies as the detection antibodies.

As shown in the following table, the detection time of the chemiluminescent method is significantly shortened compared with the limulus reagent method (50 min), and only 10min is required; the endotoxin concentration is added to 640ng/mL near the critical value, the limulus reagent method detects false positive, and the magnetic particle chemiluminescence method does not change the negative and positive detection result of the sample, so that the carboxylated magnetic bead coupled antibody eliminates the interference of endotoxin in the sample on the result.

Example 8 immunofluorescence assay based on Multi-Point mutant antifungal 1, 3-beta-D-glucan monoclonal antibodies

The embodiment provides a 1, 3-beta-D-glucan detection test strip for detecting 1, 3-beta-D-glucan of different sources, and the preparation method of the 1, 3-beta-D-glucan detection test strip comprises the following steps:

(1) Sample pad treatment:

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

(2) Preparing fluorescent microsphere marked antibody:

a. mixing the fluorescent microsphere solution with 50mmol/L MES buffer solution to obtain a fluorescent microsphere mixed solution with the fluorescent microsphere concentration of 0.1wt%, adding 10mg/mL EDC solution and 10mg/mL NHS solution into the fluorescent microsphere mixed solution, mixing, wherein the volume ratio of the fluorescent microsphere mixed solution to the EDC solution to the NHS solution is 100:1:1, carrying out shake reaction at 25 ℃ for 120min to activate the fluorescent microsphere, centrifuging at 10000g for 30min, and collecting precipitate to obtain the activated fluorescent microsphere;

b. diluting the activated fluorescent microsphere obtained in the step a by using 25mmol/L HEPES solution, adding avidin and mixing, wherein the mass ratio of the activated fluorescent microsphere to the avidin is 20:1, and carrying out oscillation reaction for 10 hours at 25 ℃ to obtain an avidin-marked fluorescent microsphere;

c. Treating the avidin-labeled fluorescent microsphere by using a blocking solution with the concentration of 1wt%, and centrifugally collecting precipitate to obtain an avidin-fluorescent microsphere compound;

d.N-hydroxysuccinimide ester activated biotin is mixed with a multipoint mutation antifungal 1, 3-beta-D-glucan monoclonal antibody according to a molar quantity ratio of 20:1, and the mixture is vibrated for 10 hours at 25 ℃ to remove unbound biotin, so that a biotin-multipoint mutation antifungal 1, 3-beta-D-glucan monoclonal antibody complex is obtained; mixing N-hydroxysuccinimide ester activated biotin and chicken IgY antibody according to the molar quantity ratio of 20:1, and oscillating for 10 hours at 25 ℃ to remove unbound biotin, thereby obtaining a biotin-chicken IgY antibody compound;

(3) Mixing the avidin-labeled fluorescent microsphere and the biotin-labeled antibody, and embedding the mixture on a fluorescent pad:

mixing the fluorescent microsphere marked avidin and the antibody marked biotin obtained in the step (2) according to the mass ratio of 10:1, wherein the mass ratio of the avidin-fluorescent microsphere complex to the biotin-multipoint mutation antifungal 1, 3-beta-D-glucan monoclonal antibody complex is 10:1, the mass ratio of the avidin-fluorescent microsphere complex to the biotin-chicken IgY antibody complex is 10:1, spraying the mixture onto a fluorescent pad according to the using amount of 5 mu L/cm and the interval of 6mm, and drying the mixture for 2h under the conditions of 37 ℃ and 25% of humidity.

(4) Coating a detection line and a quality control line on a nitrocellulose membrane:

the multi-point mutation antifungal 1, 3-beta-D-glucan monoclonal antibody and the goat anti-chicken IgY antibody are respectively streaked on a nitrocellulose membrane, the coating amount of the multi-point mutation antifungal 1, 3-beta-D-glucan monoclonal antibody is 1 mu L/cm, the coating amount of the goat anti-chicken IgY antibody is 1 mu L/cm, and the mixture is dried for 10 hours at 37 ℃ and 25% humidity.

(5) Assembling a test strip:

sequentially sticking a nitrocellulose membrane, a water absorption pad, a fluorescent pad and a sample pad on a PVC bottom plate, wherein the water absorption pad and the fluorescent pad respectively cover the nitrocellulose membrane by 2mm independently, the sample pad covers the fluorescent pad by 2mm, and the multi-point mutation antifungal 1, 3-beta-D-glucan detection test strip is obtained after assembly.

This test example uses a 1, 3-beta-D-glucan test strip to detect polysaccharide antigens extracted from two different sources of fungal strains, including antigens extracted from Candida albicans (ATCC 90028), aspergillus fumigatus (ATCC 13073).

The statistics of the sensitivity results of the reaction of the 1, 3-beta-D-glucan detection test strip with polysaccharide antigens extracted from two different fungal strains are shown in the table.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; 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 or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Binding protein comprising a 1, 3-beta-D-glucan binding domain, characterized in that the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of, or has at least 80% sequence identity with, the complementarity determining region of the amino acid sequence and has a KD of +. ^-9 Binding force of 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 complementarity determining region CDR-VL2 is X1-A-S-R-L-A-S, wherein,

x1 is G or A;

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 of claim 1, wherein the binding protein is a protein that,

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

alternatively, in the complementarity determining region CDR-VH2, X3 is A;

alternatively, in the complementarity determining region CDR-VH3, X4 is A;

alternatively, in the complementarity determining region CDR-VL1, X2 is A;

alternatively, in the complementarity determining region CDR-VL2, X1 is A;

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

3. The binding protein of claim 1, wherein the complementarity determining region comprises any one of the following combinations (a) - (i):

4. A binding protein according to any one of claims 1 to 3, wherein the binding protein comprises at least 3 CDRs; alternatively, the binding protein comprises 6 CDRs;

5. A binding protein according to any one of claims 1 to 3, wherein the binding protein comprises heavy chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4, the sequences of which are shown in SEQ ID NOs 1 to 4, and/or light chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4, the sequences of which are 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 the group consisting of the sequence of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, igD constant regions;

preferably, the constant region is of species origin of cow, 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. A biomaterial, characterized in that the biomaterial comprises any one of the following:

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

(b) A vector comprising the nucleic acid molecule of (a);

(c) A host cell comprising (a) said nucleic acid molecule or (b) said vector.

7. A method of producing a binding protein according to any one of claims 1 to 5, comprising culturing a 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 a binding protein according to any one of claims 1 to 5 for the preparation of a diagnostic agent, test strip or kit for diagnosing a fungal infection.

9.1,3-beta-D-glucan detection test strip, which is characterized in that a detection line of the test strip is obtained by scribing a binding protein according to any one of claims 1 to 5.

10. A method for detecting 1,3- β -D-glucan for non-diagnostic purposes, the method comprising a chemiluminescent assay comprising a pretreatment step of a sample to be tested, the pretreatment step comprising any one of (a) to (c):

(a) Treating a sample to be tested by alkali liquor at 37 ℃;

(b) EDTA-2Na solution is used for treating a sample to be tested at 100 ℃;

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