Plasmodium binding proteins and combinations and applications thereof

Abstract

The invention relates to a plasmodium binding protein, in particular to an anti-plasmodium monoclonal antibody, a kit comprising the antibody and related application of the anti-plasmodium monoclonal antibody to immunodetection. The anti-plasmodium monoclonal antibody can be specifically combined with histidine-rich protein-II (HRP-II) or fragments and variants thereof, and / or lactic dehydrogenase (LDH) of various plasmodium or fragments and variants thereof, shows high sensitivity and high specificity of targeting HRP-II and / or LDH, and provides a simple, rapid, sensitive and specific scheme for diagnosis of malaria.

Description

Plasmodium binding proteins and their combinations and applications Technical Field

[0001] The present invention relates to the field of immunoassays, and specifically to a malarial parasite binding protein, and more specifically to an anti-malarial parasite monoclonal antibody and an antigen-binding fragment thereof that specifically binds to malarial parasite histidine-rich protein II (HRP-II) or fragments and variants thereof, and / or lactate dehydrogenase (LDH) or fragments and variants thereof, as well as related applications of the antibody for detecting malaria. Background Art

[0002] Malaria is a life-threatening disease caused by parasites and transmitted to humans through the bites of infected female Anopheles mosquitoes. Infection occurs primarily in tropical countries. Five parasite species cause human malaria: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Plasmodium falciparum and Plasmodium vivax are the most lethal. Plasmodium falciparum is the most deadly malaria parasite and is most prevalent in Africa. Plasmodium vivax is the primary malaria parasite in most countries outside of sub-Saharan Africa.

[0003] The most common early symptoms of malaria are fever, headache, and chills, which usually begin within 10-15 days after being bitten by an infected mosquito. Malaria can cause severe symptoms such as extreme fatigue and lethargy, impaired consciousness, convulsions, difficulty breathing, dark or bloody urine, jaundice (yellowing of the eyes and skin), and abnormal bleeding, which can be life-threatening in severe cases. The gold standard for malaria diagnosis is microscopy, but malaria is mainly infected in poor and underdeveloped countries and remote areas with insufficient local equipment and limited technical personnel. In addition, the early symptoms of malaria are difficult to distinguish from other infections. This results in a delay in the early diagnosis of malaria, which is very unfavorable for the prevention and control of malaria infection.

[0004] Rapid antigen tests are based on immunological methods and are simple, rapid, intuitive, and require no complex equipment. They are recommended by the World Health Organization for malaria diagnosis. Plasmodium falciparum is unique in its production of a histidine-rich protein II (HRP-II). When the parasite infects human red blood cells, it produces large quantities of HRP-II and secretes it into the blood. Therefore, HRP-II is a specific diagnostic marker for falciparum malaria infection. However, over the past decade or so, mutant Plasmodium falciparum parasites that do not express HRP-II have emerged worldwide, leading to missed detection of falciparum malaria. Lactate dehydrogenase (LDH), expressed by all five types of Plasmodium, is another diagnostic marker for malaria infection. Using a combined test with specific antibodies targeting LDH and HRP-II not only addresses the issue of missed detection of mutant Plasmodium falciparum parasites, but also allows for the detection of Plasmodium vivax.

[0005] Therefore, there is a need in the art for a detection reagent and related method for Plasmodium with high specificity, high sensitivity and low cross-reactivity, so as to achieve a simple, rapid, sensitive and specific diagnosis of malaria.

[0006] Summary of the Invention

[0007] The inventors immunized mice with histidine-rich protein-II (HRP-II) of Plasmodium falciparum, lactate dehydrogenase (pf-LDH) protein of Plasmodium falciparum, and lactate dehydrogenase (pv-LDH) protein of Plasmodium vivax, respectively. Mouse spleen cells were fused with myeloma cells, and hybridoma cell lines that specifically bound to HRP-II or LDH were screened by ELISA. Recombinant antibodies were constructed to obtain recombinant anti-HRP-II monoclonal antibodies and recombinant anti-LDH monoclonal antibodies, respectively. Thus, the present invention was achieved.

[0008] Therefore, in the first aspect, the present invention provides an anti-malarial monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to histidine-rich protein-II (HRP-II) of malaria parasite or a fragment or variant thereof, or specifically binds to lactate dehydrogenase (LDH) of malaria parasite or a fragment or variant thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3;

[0009] In an antibody or antigen-binding fragment thereof that specifically binds lactate dehydrogenase (LDH) or a fragment or variant thereof:

[0010] V H The amino acid sequence of CDR1 is represented by X1-X2-X3-X4-X5-X6-X7, wherein X1 is G or E, X2 is Y or F, X3 is T or S, X4 is F or L, X5 is T, F or S, X6 is S, T or N, and X7 is S or Y;

[0011] V H The amino acid sequence of CDR2 is represented by X8-X9-X10-X11-X12-X13, wherein X8 is D, N, W or Y, X9 is P, S or A, X10 is S, Y or G, X11 is absent or is D, S or N, X12 is S, D or G, and X13 is F, G, D, T, N or Y;

[0012] V H The amino acid sequence of CDR3 is represented by X14-X15-G-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-Y, wherein X14 is absent or GN, X15 is absent, L or Y, X16 is S, N, D, X17 is S, N, I, X18 is absent or Y, X19 is D, L or G, X20 is E, R or Y, X21 is D, E, G or N, X22 is F, Y, T, N or G, X23 is GN, N, V, S, G, Y or H, X24 is A, T, G or S, X25 is M, L, F or I, and X26 is D or T;

[0013] V L The amino acid sequence of CDR1 is represented by X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39-X40, wherein X27 is RAS or absent, X28 is S, T, Q or K, X29 is A, V or S, X30 is S or V, X31 is A, S, E or Q, X32 is S, N or T, X33 is I, V, F or L or absent, X34 is S, T, G or L, X35 is S, Y, T or N, X36 is S, R, Y or T, X37 is R, T, K, N or absent, X38 is Y, V or S, X39 S, L or M, and X40 is H, A or absent;

[0014] V L The amino acid sequence of CDR2 is represented by X41-X42-S-X43-X44-X45-X46, wherein X41 is S, G, W or F, X42 is T or A, X43 is N or T, X44 is L or R, X45 is A, Y, C or E, and X46 is S, T or I;

[0015] V LThe amino acid sequence of CDR3 is represented by X47-X48-X49-X50-X51-X52-X53-X54-T, wherein X47 is H, G, Q or K, X48 is Q or H, X49 is Y, S or T, X50 is H, F, W or Y, X51 is R, H, N or E, X52 is S, Y, L or I, X53 is P or absent, and X54 is W, P or Y;

[0016] In an antibody or antigen-binding fragment thereof that specifically binds to histidine-rich protein-II or a fragment or variant thereof:

[0017] V H The amino acid sequence of CDR1 is represented by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 14, or SEQ ID NO: 19, or a sequence having 80% or greater, 85% or greater, 90% or greater, or 95% or greater identity thereto;

[0018] V H The amino acid sequence of CDR2 is represented by SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 15 or SEQ ID NO: 20, or a sequence having 80% or more, 85% or more, 90% or more identity thereto;

[0019] V H The amino acid sequence of CDR3 is represented by SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 21, or a sequence having greater than 80%, greater than 85%, greater than 90% or greater than 95% identity thereto;

[0020] V L The amino acid sequence of CDR1 is represented by SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:17 or SEQ ID NO:22, or a sequence having greater than 80%, greater than 85%, greater than 90% or greater than 95% identity thereto;

[0021] V L The amino acid sequence of CDR2 is represented by SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 23, or a sequence having 80% or more, 85% or more, 90% or more identity thereto; and

[0022] V LThe amino acid sequence of CDR3 is shown by SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18 or SEQ ID NO: 24, or by a sequence having greater than 80%, greater than 85%, greater than 90% or greater than 95% identity with the sequence.

[0023] In a second aspect, the present invention provides a combination of anti-Plasmodium monoclonal antibodies or antigen-binding fragments thereof, comprising:

[0024] 1) a first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 1) to 4) of the first aspect of the present invention;

[0025] 2) a first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 5) to 14) of the first aspect of the present invention; or

[0026] 3) A first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 15) to 38) of the first aspect of the present invention.

[0027] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect.

[0028] In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule of the third aspect.

[0029] In a fifth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.

[0030] In a sixth aspect, the present invention provides a method for detecting Plasmodium, comprising the step of using the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect.

[0031] In a seventh aspect, the present invention provides a method for diagnosing malaria, comprising the step of using the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect.

[0032] In an eighth aspect, the present invention provides use of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect in the preparation of a reagent for detecting Plasmodium and / or diagnosing malaria.

[0033] In a ninth aspect, the present invention provides a kit for detecting Plasmodium and / or diagnosing malaria infection, comprising: the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect; and instructions for use.

[0034] In summary, the present invention provides a new anti-malarial monoclonal antibody or an antigen-binding fragment thereof, which exhibits high sensitivity and high specificity in binding to histidine-rich protein II (HRP-Ⅱ) or lactate dehydrogenase (LDH) of the malaria parasite, can be applied to a variety of immunoassays, and can thus achieve the detection of malaria parasites at nanogram concentrations. The anti-malarial monoclonal antibody of the present invention can be used for joint detection of HRP-Ⅱ and LDH, thereby solving the problem of missed detection of mutant falciparum malaria. The anti-malarial monoclonal antibody of the present invention can also be used for joint detection of different LDHs, which can achieve the detection of falciparum malaria and vivax malaria, and then distinguish between falciparum malaria with the most serious complications and other malaria parasite infections, thereby providing a simple, rapid, sensitive and specific solution for the diagnosis of malaria, especially falciparum malaria and vivax malaria. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0036] FIG1 shows the binding reaction results of the monoclonal antibodies H1 and H2 of the present invention with histidine-rich protein-II (HRP-II) and pf-LDH protein (as a control) of Plasmodium falciparum.

[0037] FIG2 shows the binding reaction results of the recombinant monoclonal antibodies H1′-H15′ of the present invention with histidine-rich protein-II (HRP-II) and pf-LDH protein (as a control) of Plasmodium falciparum. DETAILED DESCRIPTION

[0038] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is merely illustrative of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. Furthermore, those skilled in the art will appreciate that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0040] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the subject matter.

[0041] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can generally be understood as open-ended expressions, indicating that in addition to the various elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed-ended expressions in some cases, indicating that only the various elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0042] For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood as being modified in all cases by the term "about", unless otherwise indicated. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0043] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the antibody's isotype can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 、C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The variable regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). For each heavy or light chain, the variable region comprises three CDRs, namely CDR1, CDR2 and CDR3. H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form the antigen binding site.

[0044] The rules for assigning amino acids to various regions or domains are defined in many literatures: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)); Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883; Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0045] The exact boundaries of CDRs have been defined differently according to different systems. The Kabat system not only provides a clear residue numbering system that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are called Kabat CDRs. Chothia found that certain sub-portions within the Kabat system CDRs have almost identical peptide main chain conformations despite having great diversity at the amino acid sequence level. These sub-portions are called Chothia CDRs. Chothia CDRs have boundaries that overlap with Kabat CDRs. The above-mentioned overlapping boundaries were further described by Padlan and MacCallum. The definition of CDR boundaries may not strictly adhere to the above system, such as the AbM definition. In this article, CDRs can be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al. to define CDRs. According to the Chothia numbering system, the V residues of an antibody are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 7 H CDR1 is located at positions 26 to 32, V H CDR2 is located at positions 52 to 57, V H CDR3 is located at positions 99 to 108, while V L CDR1 is located at positions 24 to 39, V L CDR2 is located at positions 55 to 61, and V LCDR3 is located at positions 94 to 102.

[0046] As used herein, the term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule can be an immunoglobulin, whether it is a natural immunoglobulin or an immunoglobulin obtained partially or entirely by a synthetic method. The antibody molecule can also include all polypeptides or proteins with an antibody binding domain, and antibody fragments with an antibody binding domain are molecules such as Fab, scFv, Fv, dAb, Fd, and bifunctional antibodies. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and they usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but can also be obtained by recombinant DNA technology (e.g., see US Patent 4,816,567). As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by Ala or A, glycine can be represented by Gly or G, valine can be represented by Val or V, leucine can be represented by Leu or L, isoleucine can be represented by Ile or I, proline can be represented by Pro or P, phenylalanine can be represented by Phe or F, tyrosine can be represented by Tyr or Y, tryptophan can be represented by Trp or W, serine can be represented by Ser or S, threonine can be represented by Thr or T, cysteine ​​can be represented by Cys or C, methionine can be represented by Met or M, asparagine can be represented by Asn or N, glutamine can be represented by Gln or Q, aspartic acid can be represented by Asp or D, glutamic acid can be represented by Glu or E, lysine can be represented by Lys or K, arginine can be represented by Arg or R, and histidine can be represented by His or H.

[0047] As used herein, the term "recombinant antibody" refers to an antibody obtained by cloning an antibody gene into an expression vector and then transfecting the expression vector into a suitable host cell line for expression by molecular biology techniques. The recombinant antibody encoding gene may be identical or non-identical to the antibody encoding gene of natural origin. For example, the complete encoding gene of the antibody obtained by immune animal can be cloned into an expression vector for expression, thereby obtaining an antibody identical to the antibody obtained by immune animal, or the gene encoding the variable region (including heavy chain variable region and light chain variable region) of the antibody obtained by immune animal can be cloned into an expression vector together with the gene encoding the constant region of the antibody in another species (such as human) source for expression, thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies with mouse heavy chain and light chain variable regions connected to human constant regions. The antibody is commonly referred to as a "chimeric antibody" in the art.

[0048] As used herein, the term "antigen-binding fragment" refers to fragments and antibody analogs from antibodies that are capable of binding to an antigen, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen-binding fragment retains at least some of the binding activity of the parental antibody. Typically, when the activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Among them, a "Fab fragment" consists of a light chain, the CH1 of a heavy chain, and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" contains one light chain and part of one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); thus, an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule. An "Fv region" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0049] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0050] In the present invention, primers can also be used to perform PCR amplification on the nucleotide sequence encoding the antibody. In the primer sequence, some sites only involve a single base, such as any one of adenine (A), guanine (G), cytosine (C) and thymine (T), while some sites involve a combination of two, three or four bases. In this case, these bases are referred to as degenerate bases of each other, which are mainly determined based on the degeneracy of the codons. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, H represents A / T / C, B represents G / T / C, V represents G / A / C, D represents G / A / T, and N represents A / T / C / G.

[0051] As used herein, the terms sequence "identity," "identity," or "homology" have their art-recognized meanings, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art to apply conservative amino acid substitutions in peptides or proteins, and generally can be made without altering the biological activity of the resulting molecule. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0052] As previously described, the present invention aims to provide a highly sensitive and specific anti-malarial monoclonal antibody. The inventors immunized mice with histidine-rich protein-II (HRP-II) and lactate dehydrogenase (pf-LDH) proteins from Plasmodium falciparum, and lactate dehydrogenase (pv-LDH) protein from Plasmodium vivax, respectively. The spleen cells from the mice were fused with myeloma cells, and hybridoma cell lines that specifically bound to Plasmodium HRP-II or LDH proteins were screened by ELISA, thereby completing the present invention.

[0053] Therefore, in the first aspect, the present invention provides an anti-malarial monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to histidine-rich protein-II (HRP-II) of malaria parasite or a fragment or variant thereof, or specifically binds to lactate dehydrogenase (LDH) of malaria parasite or a fragment or variant thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3;

[0054] In an antibody or antigen-binding fragment thereof that specifically binds to lactate dehydrogenase or a fragment or variant thereof:

[0055] V H The amino acid sequence of CDR1 is represented by X1-X2-X3-X4-X5-X6-X7, wherein X1 is G or E, X2 is Y or F, X3 is T or S, X4 is F or L, X5 is T, F or S, X6 is S, T or N, and X7 is S or Y;

[0056] V H The amino acid sequence of CDR2 is represented by X8-X9-X10-X11-X12-X13, wherein X8 is D, N, W or Y, X9 is P, S or A, X10 is S, Y or G, X11 is absent or is D, S or N, X12 is S, D or G, and X13 is F, G, D, T, N or Y;

[0057] V H The amino acid sequence of CDR3 is represented by X14-X15-G-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-Y, wherein X14 is absent or GN, X15 is absent, L or Y, X16 is S, N, D, X17 is S, N, I, X18 is absent or Y, X19 is D, L or G, X20 is E, R or Y, X21 is D, E, G or N, X22 is F, Y, T, N or G, X23 is GN, N, V, S, G, Y or H, X24 is A, T, G or S, X25 is M, L, F or I, and X26 is D or T;

[0058] V LThe amino acid sequence of CDR1 is represented by X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39-X40, wherein X27 is RAS or absent, X28 is S, T, Q or K, X29 is A, V or S, X30 is S or V, X31 is A, S, E or Q, X32 is S, N or T, X33 is I, V, F or L or absent, X34 is S, T, G or L, X35 is S, Y, T or N, X36 is S, R, Y or T, X37 is R, T, K, N or absent, X38 is Y, V or S, X39 S, L or M, and X40 is H, A or absent;

[0059] V L The amino acid sequence of CDR2 is represented by X41-X42-S-X43-X44-X45-X46, wherein X41 is S, G, W or F, X42 is T or A, X43 is N or T, X44 is L or R, X45 is A, Y, C or E, and X46 is S, T or I;

[0060] V L The amino acid sequence of CDR3 is represented by X47-X48-X49-X50-X51-X52-X53-X54-T, wherein X47 is H, G, Q or K, X48 is Q or H, X49 is Y, S or T, X50 is H, F, W or Y, X51 is R, H, N or E, X52 is S, Y, L or I, X53 is P or absent, and X54 is W, P or Y;

[0061] In an antibody or antigen-binding fragment thereof that specifically binds to histidine-rich protein-II or a fragment or variant thereof:

[0062] V H The amino acid sequence of CDR1 is represented by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 14 or SEQ ID NO: 19, or a sequence having 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto;

[0063] V HThe amino acid sequence of CDR2 is set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:15 or SEQ ID NO:20, or a sequence having 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto;

[0064] V H The amino acid sequence of CDR3 is represented by SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:21, or a sequence having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto;

[0065] V L The amino acid sequence of CDR1 is represented by SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:17 or SEQ ID NO:22, or a sequence having 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto;

[0066] V L The amino acid sequence of CDR2 is represented by SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:23, or a sequence having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto; and

[0067] V LThe amino acid sequence of CDR3 is shown by SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18 or SEQ ID NO:24, or by a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto.

[0068] In a specific embodiment, the anti-Plasmodium monoclonal antibody of the present invention can be an anti-HRP-II monoclonal antibody or an anti-LDH monoclonal antibody.

[0069] In the context of the present invention, the terms "anti-HRP-II monoclonal antibody", "anti-HRP-II antibody" and "HRP-II antibody" have the same meaning and are intended to refer to an isolated antibody that specifically binds to HRP-II protein or a fragment or variant thereof.

[0070] In the context of the present invention, the terms "anti-LDH monoclonal antibody", "anti-LDH antibody" and "LDH antibody" have the same meaning and are intended to refer to an isolated antibody that specifically binds to LDH protein or a fragment or variant thereof.

[0071] In the context of the present invention, the term "lactate dehydrogenase (LDH)" can be divided into lactate dehydrogenase derived from Plasmodium falciparum (pf-LDH), lactate dehydrogenase derived from Plasmodium vivax (pv-LDH), lactate dehydrogenase derived from Plasmodium malariae (pm-LDH) and lactate dehydrogenase derived from Plasmodium ovale (po-LDH) according to their sources.

[0072] The lactate dehydrogenase (pf-LDH) enzyme in Plasmodium falciparum is essential for the anaerobic life cycle of the Plasmodium parasite, and compounds that inhibit this enzyme also kill the parasite. The pf-LDH assay is one of the in vitro methods used to assess anti-plasmodial activity. The amino acid sequence of pf-LDH is, for example, SEQ ID NO:105 shows: MAPKAKIVLVGSGMIGGVMATLIVQKNLGDVVLFDIVKNMPHGKALDTSHTNVMAYSNCKVSGSNTYDDLAGADVVIVTAGFTKAPGKSDKEWNNRDDLLPLNNKIMIEIGHIKKNCPNAFIIVVTNPVDVMVQLSHQHSGVPKNKIIGLGGV LDTSRLKYYISQKLNVCPRDVNAHIVGAHGNKMVPLKRYITVGGIPLQEFINNKLISDAELEAIFDRTVNTALEIVNLHASPYVAPAAAIIEMAESYLKDLKKVLICSTLLEGQYGHSDIFGGTPVVLGANGVEQVIELQLNSEEKAKFDEAIAETKRMKALA

[0073] Lactate dehydrogenase (pv-LDH) in Plasmodium vivax is an essential enzyme in the glycolysis pathway and is required for the development and reproduction of P. vivax. Therefore, pv-LDH can be used as a biomarker for the diagnosis of P. vivax malaria and a potential molecular target for the development of antimalarial drugs. The amino acid sequence of pv-LDH is, for example, SEQ ID NO:106 shows: MTPKPKIVLVGSGMIGGVMATLIVQKNLGDVVMFDVVKNMPQGKALDTSHSNVMAYSNCKVTGSNSYDDLKGADVVIVTAEFTKAPGKSVKEWNNRDDLLPLNNKIMIEEGHIKNLCPNTFIIVVTNPVDVMVQLLFEHSGVPKNKIIGLGGV LDTSRLKYYISQKLNVCPRDVNALIVGAHGNKMVLLKRYITVGGIPLQEFINNKKITDEEVEGIFDRTVNTALEIVNLLASPYVAPAAAIIEMAESYLKDIKKVLVCSTLLEGQYGHSNIFGGTPLVIGGTGVEQVIELQLNAEEKTKFDEAVAETKRKKALI

[0074] The amino acid sequence of lactate dehydrogenase (pm-LDH) in Plasmodium malariae is shown, for example, as SEQ ID NO: 107: VLVGSGMIGGVMATLIVQKNLGDVVMFDIVKNMPYGKALDTSHMNVMAYSNCKVTGSNSYEDLKGADVVIVTAGFTKVPGKSDKEWNRDDLLPLNNKIMIEIGGHVKNYCPNAFIIVVTNPVDVMVQLLHKHSGVPKNKIVGLGGVLDTSRLKYYISQKLNVCPRDVNALIVAAHGNKMVPLKRYITVGGIPLQEFINNKKITDAELDAIFDRTVNTALEIVNLHASPYVAPAAAIIEMAESYIKDLKKVLICSTLLEGQYGHSDIFGGTPLVLGANGVEQVIELQLNSEEKKKFDEAI.

[0075] The amino acid sequence of lactate dehydrogenase (po-LDH) in Plasmodium ovale is shown, for example, in SEQ ID NO: 108: IVLVGSGMIGGLMATLIVQKNLGDVVMFDVVKNMPQGKALDTSHSNVMAYSNCKVTGSNSYDDLKGADVVIVTAGFTKAPGKSDKEWNRDDLLPLNNKIMIEIGGHIKNLCPNAFIIVVTNPVDVMVQLLFEHSGVPKNKIIGLGGVLDTSRLKYYISQKLNVCPRDVNALIVGAHGNKMVLLKRYITVGGIPLQEFINNKKITDEEVEGIFDRTVNTALEIVNLLASPYVAPAAAIIEMAESYLKDIKKVLVCSTLLEGQYGHSNIFGGTPLVIGGTGVEQVIELQLNAEEKTKFDEAVAETKRMKALI.

[0076] Since the homology of the four Plasmodium lactate dehydrogenases (pf-LDH, pv-LDH, pm-LDH, and po-LDH) is greater than 86%, the anti-LDH monoclonal antibodies of the present invention can specifically bind to one, two, three, or four of these four LDHs or their fragments or variants.

[0077] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 33-35, H CDR1, V H CDR2 and V HCDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 36-38, respectively. L CDR1, V L CDR2 and V L CDR3.

[0078] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 39-41, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0079] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44, and SEQ ID NO: 41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0080] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0081] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 49-51, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 52-54, respectively. L CDR1, V L CDR2 and V L CDR3.

[0082] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 55-57, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L CDR3.

[0083] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 60-62, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 53 and SEQ ID NO: 64, respectively. L CDR1, V L CDR2 and V L CDR3.

[0084] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 56 and SEQ ID NO: 65, respectively. H CDR1, V H CDR2 and V HCDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 53 and SEQ ID NO: 67, respectively. L CDR1, V L CDR2 and V L CDR3.

[0085] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 68, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 69 and SEQ ID NO: 70, respectively. L CDR1, V L CDR2 and V L CDR3.

[0086] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 71-73, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3.

[0087] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 77 and SEQ ID NO: 73, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 78, SEQ ID NO: 75 and SEQ ID NO: 79, respectively. L CDR1, V L CDR2 and V L CDR3.

[0088] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 80 and SEQ ID NO: 81, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 82-84, respectively. L CDR1, V L CDR2 and V L CDR3.

[0089] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56, and SEQ ID NO: 86, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3.

[0090] In another specific embodiment, the heavy chain variable region of the anti-LDH monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56, and SEQ ID NO: 88, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3.

[0091] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises heavy chain complementary determining regions V and V represented by amino acid sequences of SEQ ID NOs: 1-3, respectively. H CDR1, V H CDR2 and V HCDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively. L CDR1, V L CDR2 and V L CDR3.

[0092] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3.

[0093] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3.

[0094] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3.

[0095] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2, and SEQ ID NO: 13, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3.

[0096] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 14-16, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 22-24, respectively. L CDR1, V L CDR2 and V L CDR3.

[0097] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2, and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively. L CDR1, V L CDR2 and V L CDR3.

[0098] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V HCDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0099] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 2, and SEQ ID NO: 16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3.

[0100] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3.

[0101] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and VL CDR3.

[0102] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0103] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2, and SEQ ID NO: 13, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3.

[0104] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 14-16, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3.

[0105] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 16, respectively.H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0106] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 2, and SEQ ID NO: 21, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3.

[0107] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 21, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 24, respectively. L CDR1, V L CDR2 and V L CDR3.

[0108] In another specific embodiment, the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 20, and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 18, respectively.L CDR1, V L CDR2 and V L CDR3.

[0109] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 8, and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3.

[0110] In another specific embodiment, the heavy chain variable region of the anti-HRP-II monoclonal antibody comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 19-21, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 22-24, respectively. L CDR1, V L CDR2 and V L CDR3.

[0111] In a specific embodiment, the antibody is a complete antibody comprising a variable region (V region) and a constant region (C region). For the antibodies of the present invention, any framework region (FR) and any constant region can be used. The amino acid sequence of the FR or constant region used in the antibodies of the present invention can be the amino acid sequence of the original FR or constant region as a source, or it can be a different amino acid sequence obtained by performing one or more amino acid substitutions, deletions, additions and / or insertions on the amino acid sequence of the original FR or constant region. The structure used to support the CDR or CDR group of the present invention generally belongs to an antibody heavy chain or light chain sequence or a major part thereof, wherein the CDR or CDR group is located in a region corresponding to a naturally occurring V region encoded by a rearranged immunoglobulin gene. H and V L The CDR or CDR group of the antibody variable domain corresponds to the position of the antibody variable domain.

[0112] As an example, each framework region (FR) may have the following sequence:

[0113] HFR1 (heavy chain framework region 1): QIQLVQSGPELKKPGETVKISCKAS (SEQ ID NO: 25);

[0114] HFR2 (heavy chain framework region 2): GMDWVKQAPGKGLKWMGWI (SEQ ID NO: 26);

[0115] HFR3 (heavy chain framework region 3): TYSDDFEGRFAFSLETSASTVYLQINNLKNADTATYFCAR (SEQ ID NO: 27);

[0116] HFR4 (heavy chain framework region 4): WGQGTLVTVS (SEQ ID NO: 28);

[0117] LFR1 (light chain framework region 1): NVVLTQTPQFLLVSTGDRVTITC (SEQ ID NO: 29);

[0118] LFR2 (light chain framework region 2): WYQQKPGQSPKLLVF (SEQ ID NO: 30);

[0119] LFR3 (light chain framework region 3): GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC (SEQ ID NO: 31);

[0120] LFR4 (light chain framework region 4): FGGGTKLDIK (SEQ ID NO: 32).

[0121] As yet another example, each framework region (FR) may also have the following sequence:

[0122] HFR1: QVQLQQSGAELVKPGASVKLSCKAS (SEQ ID NO:89);

[0123] HFR2: YINWIKQRPGQGLEWIGEI (SEQ ID NO:90);

[0124] HFR3: TNFNEKFKSKATLSVDKSSSTAYMQLSSLTSEDSAVYYCIR (SEQ ID NO:91);

[0125] HFR4: WGQGTLVTVSA (SEQ ID NO:92);

[0126] LFR1: DAVMTQIPLSLPVSLGDQVSISC (SEQ ID NO:93);

[0127] LFR2: WYLQKPGQSPQLLIY (SEQ ID NO:94);

[0128] LFR3: GVLDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:95);

[0129] LFR4: FGSGTKLEIK (SEQ ID NO:96).

[0130] As yet another example, each framework region (FR) may also have the following sequence:

[0131] HFR1: EVQLQQSGPEVVKPGASVKISCKAS (SEQ ID NO:97);

[0132] HFR2: WINWVKQRPGHGLEWIGDI(SEQ ID NO:98);

[0133] HFR3: TNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR (SEQ ID NO:99);

[0134] HFR4: WGQGTTLTVSS (SEQ ID NO: 100);

[0135] LFR1: DIVMSQSPSSLAVSAGEKVTMSC (SEQ ID NO:101);

[0136] LFR2: WYQQKPGQSPKLLIY(SEQ ID NO:102);

[0137] LFR3: GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEQ ID NO: 103);

[0138] LFR4: FGGGTKLEIK (SEQ ID NO: 104).

[0139] In a specific embodiment, the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, and the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4. It will be appreciated by those skilled in the art that the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 are closely related to the heavy chain complementary determining regions V H CDR1, V H CDR2 and V HCDR3 is arranged from amino terminus to carboxyl terminus according to HFR1, V H CDR1、HFR2、V H CDR2、HFR3、V H The sequence of CDR3 and HFR4 constitutes the heavy chain variable region sequence; the light chain framework region LFR1, LFR2, LFR3 and LFR4 and the light chain complementary determining region V L CDR1, V L CDR2 and V L CDR3 is arranged from amino terminus to carboxyl terminus according to LFR1, V L CDR1, LFR2, V L CDR2, LFR3, V L The sequential arrangement of CDR3 and LFR4 constitutes the light chain variable region sequence.

[0140] In a specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 have the sequences shown in SEQ ID NOs: 25-28, respectively, or sequences having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto; the light chain framework regions LFR1, LFR2, LFR3 and LFR4 have the sequences shown in SEQ ID NOs: 25-28, respectively. The sequences shown in NO:29-32, or sequences having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto.

[0141] In another specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown in SEQ ID NOs: 89-92, or sequences having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto; the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown in SEQ ID NOs: 89-92, or sequences having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto; The sequences shown in NO:93-96, or sequences having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even greater than 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto.

[0142] In another specific embodiment, the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown in SEQ ID NOs: 97-100, or sequences having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto; the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown in SEQ ID NOs: 97-100, or sequences having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto; The sequences shown in NO:101-104, or sequences having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto.

[0143] In another specific embodiment, the antibody further includes a constant region sequence, such as but not limited to a constant region sequence selected from any one of IgG, IgA, IgM, IgE and IgD. Those skilled in the art can make a selection based on needs, and this is not particularly limited herein.

[0144] In another specific embodiment, the species origin of the constant region sequence can be rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human, but is not limited thereto.

[0145] In a specific embodiment, the anti-Plasmodium monoclonal antibody of the present invention can be a murine antibody, a chimeric antibody, or a humanized antibody. For example, the anti-Plasmodium monoclonal antibody of the present invention can be a chimeric antibody formed by splicing the variable region of a murine antibody with the constant region of a human antibody.

[0146] In a specific embodiment, the constant region of the antibody of the present invention is derived from mouse, that is, the anti-Plasmodium monoclonal antibody of the present invention is a murine antibody.

[0147] In some embodiments, the anti-malarial antibodies of the present invention can specifically bind to LDH or a fragment or variant thereof or specifically bind to HRP-II or a fragment or variant thereof and have a specific affinity of at least 2.5×10 -12 M, at least 2.7×10 -12 M, 2.9×10 -12 M, 3.0×10 -12 M, 3.2×10 -12 M, 3.4×10 -12 M, 3.6×10 -12 M, 3.8×10 -12 M, 4.0×10 -12 M, 4.2×10 -12 M, 4.4×10 -12 M, 4.6×10 -12 M, 4.8×10 -12 M, 5.0×10 -12 M, 6.0×10 -12 M, 7.0×10 -12 M, 8.0×10 -12 M, 9.0×10 -12 M, 10×10 -12 M, 15×10 -12 M, 20×10 -12 M, 40×10 -12 M, 60×10 -12 M, 80×10 -12 M, 100×10 -12 M, 150×10 -12 M, 200×10 -12 M, 300×10 -12 M, 400×10 -12 M, 500×10 -12 M, 600×10 -12 M, 700×10 -12 M, 800×10 -12 M, 900×10 -12 M, 1000×10 -12 M, 1500×10 -12 M, 2000×10 -12 M, 2500×10 -12 M, 3000×10 -12 M, 3500×10 -12M, 4000×10 -12 M, 4500×10 -12 M, 5000×10 -12 EC of M 50 .

[0148] In a second aspect, the present invention provides a combination of anti-Plasmodium monoclonal antibodies or antigen-binding fragments thereof, comprising:

[0149] 1) a first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 1) to 4) of the first aspect of the present invention;

[0150] 2) a first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 5) to 14) of the first aspect of the present invention; or

[0151] 3) A first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 15) to 38) of the first aspect of the present invention.

[0152] In the context of the present invention, the terms "first" and "second" in the expressions "first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof", "second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof", "first antibody" and "second antibody" are for the purpose of distinction only and are not intended to limit the order, rank, etc.

[0153] In a preferred embodiment, the first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region comprising heavy chain complementary determining regions V represented by amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46, and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L The second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region including a heavy chain complementary determining region V represented by an amino acid sequence of SEQ ID NOs: 39-41, H CDR1, V H CDR2 and V HThe light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3 antibodies or antigen-binding fragments thereof.

[0154] In a preferred embodiment, the first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region comprising heavy chain complementary determining regions V and V represented by amino acid sequences of SEQ ID NOs: 39-41, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L The second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region including a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44, and SEQ ID NO: 41, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3 antibodies or antigen-binding fragments thereof.

[0155] In a preferred embodiment, the first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region comprising heavy chain complementary determining regions V and V represented by amino acid sequences of SEQ ID NOs: 49-51, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 and the light chain variable region V CDR3 including ... L CDR1, V L CDR2 and V LThe second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region including a heavy chain complementary determining region V represented by amino acid sequences of SEQ ID NOs: 71-73, H CDR1, V H CDR2 and V H The CDR3 and the light chain variable region thereof include the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3 antibodies or antigen-binding fragments thereof.

[0156] In a preferred embodiment, the first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region comprising heavy chain complementary determining regions V and V represented by amino acid sequences of SEQ ID NOs: 55-57, respectively. H CDR1, V H CDR2 and V H The CDR3 and the light chain variable region thereof include the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L The second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56, and SEQ ID NO: 86, respectively. H CDR1, V H CDR2 and V H The CDR3 and the light chain variable region thereof include the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3 antibodies or antigen-binding fragments thereof.

[0157] In the present invention, the combined use of the anti-malarial monoclonal antibodies can achieve pan-type detection and typing detection of malaria parasites. For example, the combination of recombinant antibodies L1' and L2' and the combination of L5' and L10' can bind to four lactate dehydrogenases (pf-LDH, pv-LDH, pm-LDH, po-LDH), thereby providing a universal malaria parasite detection method. For another example, the combination of recombinant antibodies L2' and L4' can only bind to the pv-LDH antigen, and has no reaction with pf-LDH, pm-LDH, po-LDH and HRP-Ⅱ, so typing detection of Plasmodium vivax can be achieved. For another example, the combination of recombinant antibodies L6' and L13' can bind to the pf-LDH antigen, but has no reaction to pv-LDH, pm-LDH and po-LDH, thereby detecting Plasmodium falciparum and mutant Plasmodium falciparum. Therefore, without wishing to be bound by theory, by selecting different antibodies of the present invention for combination, it is possible to specifically bind to one, two, three or four of the four LDHs or fragments or variants thereof.

[0158] In the present invention, the anti-Plasmodium monoclonal antibody can be used as a coating antibody. For example, the anti-Plasmodium monoclonal antibody can be bound to a solid phase, such as a solid support. There are no particular limitations on the solid support used in the present invention, and it can be a porous or non-porous material, such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, and the like.

[0159] The anti-malarial monoclonal antibodies of the present invention can also be used as labeled antibodies. For example, the anti-malarial monoclonal antibodies can be combined with magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, etc.

[0160] It is also not expected to be bound by theory, and the skilled person will understand that the specific antibody combination listed above is merely an exemplary or preferred antibody combination. Those skilled in the art can screen out antibody combinations with different specificities based on the well-known means of the prior art to carry out typing or general detection of Plasmodium. It is also understandable that the first anti-Plasmodium monoclonal antibody and the second anti-Plasmodium monoclonal antibody used in the above-mentioned detection are preferably directed against the same immunogen, for example, the first anti-Plasmodium monoclonal antibody is an anti-pf-LDH antibody, and the second anti-Plasmodium monoclonal antibody is also an anti-pf-LDH antibody. For another example, the first anti-Plasmodium monoclonal antibody is an anti-pv-LDH antibody, and the second anti-Plasmodium monoclonal antibody is also an anti-pv-LDH antibody.

[0161] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect.

[0162] For those skilled in the art, knowing the amino acid sequence of a protein, such as the anti-Plasmodium monoclonal antibody or binding fragment thereof of the present invention, it is well within their capabilities to determine its nucleic acid encoding sequence. Furthermore, to obtain a monoclonal antibody by recombinant means, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into an expression cell, which can then express the antibody protein.

[0163] In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule of the third aspect.

[0164] In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

[0165] In a specific embodiment, the vector may be a pTOPO vector.

[0166] In another specific embodiment, the vector can be a eukaryotic expression vector.

[0167] In a preferred embodiment, the pcDNA vector may be pCDNA3.1.

[0168] In a fifth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.

[0169] The expression cell is prepared by introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into a host cell by molecular biological methods well known to those skilled in the art.

[0170] As previously described, the present inventors immunized mice with HRP-II, pf-LDH, and pv-LDH proteins, respectively. Mouse spleen cells were then fused with myeloma cells and screened by ELISA to identify hybridoma cell lines that specifically bind to HRP-II or LDH. After identifying monoclonal cell lines secreting the target antibody, heavy and light chain variable region cDNAs can be recovered from the cell lines by RT-PCR, and appropriate immunoglobulin constant regions (e.g., human constant regions) can be selected. These heavy and light chain variable region cDNAs and constant region cDNAs are then transformed into host cells, such as COS or CHO cells, to produce cells expressing the target antibody of the present invention. Using monoclonal antibodies and other antibodies and recombinant DNA techniques, other antibodies or chimeric molecules that retain the specificity of the original antibody can be generated. These techniques may include introducing DNA encoding the immunoglobulin variable region or complementarity determining region (CDR) of an antibody into the constant region, or constant region plus framework regions, of a different immunoglobulin.

[0171] In a specific embodiment, the expressing cells can be mammalian cells, such as Chinese hamster ovary cells, baby hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. In a more specific embodiment, the expressing cells can be, for example, monkey kidney cells transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse thymoma cells (NSO), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (HepG2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but are not limited thereto.

[0172] In a sixth aspect, the present invention provides a method for detecting Plasmodium, comprising the step of using the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect.

[0173] Those skilled in the art will appreciate that the detection methods may be used for non-diagnostic purposes or for diagnostic purposes.

[0174] In a specific embodiment, the malarial parasite includes any one or more of Plasmodium falciparum, mutant Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale.

[0175] The method for detecting malarial parasites of the present invention is achieved by the anti-HRP-II monoclonal antibody and the anti-LDH monoclonal antibody of the present invention specifically binding to HRP-II or LDH of malarial parasites.

[0176] As previously mentioned, the anti-LDH monoclonal antibodies of the present invention can specifically bind to one of the four types of LDH, namely pf-LDH, pv-LDH, pm-LDH, and po-LDH, i.e., they are typing-specific. However, without wishing to be bound by theory, the anti-LDH monoclonal antibodies of the present invention may also bind to two, three, or four of the four types of LDH. Antibodies that can bind to all four types of LDH are called pan-LDH antibodies. Pan-LDH antibodies can thus be used to simultaneously detect pf-LDH, pv-LDH, pm-LDH, and po-LDH in a sample. Thus, detection of different LDHs can be achieved based on the anti-LDH monoclonal antibodies of the present invention. For example, LDH antibodies that specifically bind to pf-LDH or pv-LDH can be used to detect Plasmodium falciparum or Plasmodium vivax antigens, or a combination of these two antibodies can be used to type Plasmodium falciparum and Plasmodium vivax in a sample. Anti-LDH antibodies can also be combined with other antibodies, such as anti-HRP-II antibodies, to perform joint detection, thereby detecting or typing Plasmodium falciparum antigens.

[0177] In a specific embodiment, the following antibody combination is used to detect all of Plasmodium falciparum, mutant Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale:

[0178] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 39-41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; or

[0179] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 49-51, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 52-54, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 71-73, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3.

[0180] In a further embodiment, the combination of recombinant antibodies L1' and L2' or the combination of L5' and L10' is used to detect all of Plasmodium falciparum, mutant Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale.

[0181] In yet another specific embodiment, the following antibody combination is used to detect Plasmodium vivax:

[0182] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 39-41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: and SEQ ID NO: H CDR1, V HCDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO:, SEQ ID NO:37 and SEQ ID NO:43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0183] In a further embodiment, a combination of recombinant antibodies L2' and L4' is used to detect Plasmodium vivax.

[0184] In yet another specific embodiment, the following antibody combination is used to detect Plasmodium falciparum or mutant Plasmodium falciparum:

[0185] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 55-57, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, whose heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 86, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3.

[0186] In a further embodiment, a combination of recombinant antibodies L6' and L13' is used to detect Plasmodium falciparum or mutant Plasmodium falciparum.

[0187] The anti-LDH monoclonal antibody of the present invention can detect LDH (or LDH fragment) levels in a sample of at least 0.05 ng / mL, 0.06 ng / mL, 0.07 ng / mL, 0.08 ng / mL, 0.09 ng / mL, 0.10 ng / mL, 0.11 ng / mL, 0.12 ng / mL, 0.13 ng / mL, 0.14 ng / mL, 0.15 ng / mL, 0.16 ng / mL, 0.17 ng / mL, 0.18 ng / mL, 0.19 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 7ng / mL, 0.18ng / mL, 0.19ng / mL, 0.20ng / mL, 0.25ng / mL, 0.30ng / mL, 0.35ng / mL, 0.40ng / mL, 0.4 5ng / mL, 0.50ng / mL, 0.55ng / mL, 1ng / mL, 5ng / mL, 10ng / mL, 15ng / mL, 20ng / mL, 25ng / mL or 30ng / mL.

[0188] In a specific embodiment, the detection is performed by any one or more of immunochromatography, enzyme-linked antibody assay (ELISA), chemiluminescence, and electrochemiluminescence.

[0189] In yet another specific embodiment, the detection can be a direct method, an indirect method, a sandwich method, or a competitive method.

[0190] For example, the specific steps of the detection may include: (1) obtaining a biological sample; (2) contacting the biological sample with the anti-malarial monoclonal antibody of the present invention to form an antibody-HRP-II or antibody-LDH complex; (3) contacting the complex with an antibody with a detection label; (4) determining the presence, amount or type of HRP-II or LDH in the sample by detecting the signal of the label.

[0191] In a preferred embodiment, the immunochromatography includes but is not limited to fluorescent microsphere immunochromatography, colloidal gold immunochromatography, colored latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography and quantum dot immunochromatography.

[0192] In the present invention, the anti-Plasmodium monoclonal antibody can be used as a coating antibody. For example, the anti-Plasmodium monoclonal antibody is bound to a solid phase, such as a solid support. The solid support used in the detection method of the present invention is not particularly limited and can be a porous or non-porous material, such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, etc.

[0193] Without wishing to be bound by theory, the anti-Plasmodium monoclonal antibodies of the present invention may also be used as labeled antibodies. For example, the anti-Plasmodium monoclonal antibodies may be bound to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, and the like.

[0194] For example, when performing colloidal gold immunochromatography test, the anti-malarial monoclonal antibody of the present invention can be labeled with colloidal gold, and another anti-malarial antibody is coated with nitrocellulose membrane (NC membrane), and the membrane is drawn to obtain a detection line (T line). According to the immune test strip preparation method, the colloidal gold test paper is assembled. During detection, the analyte in the positive sample is combined with the anti-malarial antibody labeled with colloidal gold to form a complex, and the complex is combined with the coated antibody at the T line to form a sandwich complex, where the colloidal gold aggregation precipitation shows red, indicating that the sample is positive.

[0195] For another example, when performing an immunochromatographic test based on colored latex microspheres, a rapid immunochromatographic test card can be assembled in a conventional manner using latex microspheres labeled with the anti-Plasmodium monoclonal antibody of the present invention, a nitrocellulose membrane (NC membrane) coated with another anti-Plasmodium antibody, a sample pad, absorbent paper, a polyester plate, etc. During the test, the analyte in the positive sample binds to the latex microspheres labeled with the monoclonal anti-Plasmodium antibody, and an agglutination reaction occurs at room temperature. After standing for a period of time, the result can be observed and determined by the naked eye.

[0196] For another example, when performing a fluorescent microsphere immunochromatographic test, the time-resolved fluorescent microspheres can be labeled with the anti-malarial monoclonal antibody of the present invention, and a nitrocellulose membrane (NC membrane) can be coated with another anti-malarial antibody, and assembled with a sample pad to form an immunochromatographic rapid test card. During testing, the analyte in the sample binds to the fluorescent microsphere-labeled antibody in the conjugate pad and chromatographs forward through capillary action. After reaching the detection area, it further binds to another monoclonal anti-malarial antibody fixed on the detection line T line to form a double-antibody sandwich. After the chromatography is completed, the fluorescence intensity of the T line and the C line is read with an immunofluorescence instrument and the T / C value is calculated. The content of the analyte in the sample can be calculated using the instrument's built-in standard curve.

[0197] The present inventors have found that the anti-malarial monoclonal antibodies of the present invention, whether used as coated antibodies or labeled antibodies, exhibit extremely high detection sensitivity. For example, the sensitivity of malaria parasite detection using colloidal gold immunochromatography can reach a concentration of 0.1 ng / mL or lower.

[0198] The inventors have further discovered that in the detection of the present invention, the labeled antibody and the coating antibody can be the same or different, and preferably different. That is, when the anti-malarial monoclonal antibody of the present invention is used for immunochromatography to detect Plasmodium, it can be used as a labeled antibody to label magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, colloidal gold, etc., and can also be used as a coating antibody to coat solid supports such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, etc.

[0199] In yet another specific embodiment, the detection can be a quantitative detection.

[0200] For example, quantitative detection can be performed using a sandwich ELISA. Sandwich ELISA involves at least two antibodies: at least one capture antibody (primary antibody) and at least one detection antibody (secondary antibody). The amount of antigen bound by the two antibodies is measured. The at least two antibodies form an immune complex with the antigen called a "sandwich." More specifically, the sandwich ELISA may include the following steps: (1) contacting the sample with a first antibody, which binds to HRP-II, an HRP-II fragment, LDH, or an LDH fragment to form a first antibody-HRP-II complex or a first antibody-LDH complex; (2) contacting the first antibody-HRP-II complex or the first antibody-LDH complex with at least one second antibody, which binds to an epitope on HRP-II, an HRP-II fragment, LDH, or an LDH fragment that is not bound by the first antibody to form a first antibody-HRP-II-second antibody complex or a first antibody-LDH-second antibody complex, wherein the first antibody or the second antibody includes a detectable label; (3) determining the amount of HRP-II, an HRP-II fragment, LDH, or an LDH fragment present in the test sample or the type of LDH or LDH fragment based on the signal generated by the detectable label in the first antibody-HRP-II-second antibody complex or the first antibody-LDH-second antibody complex.

[0201] In a specific embodiment, the first antibody and the second antibody can be anti-HRP-II antibodies.

[0202] In another specific embodiment, the first antibody and the second antibody can be anti-LDH monoclonal antibodies. Those skilled in the art will appreciate that the first and second antibodies used in the above-mentioned detection are preferably directed against the same LDH immunogen. For example, the first antibody is an anti-pf-LDH antibody, and the second antibody is also an anti-pf-LDH antibody. For another example, the first antibody is an anti-pv-LDH antibody, and the second antibody is also an anti-pv-LDH antibody.

[0203] In another specific embodiment, the detection can be achieved by labeling HRP-II or its fragment or variant, and / or LDH or its fragment or variant of Plasmodium by using a signal-displaying indicator.

[0204] In a further specific embodiment, the signal displayable indicator can be selected from, but not limited to, any one of colloidal gold, fluorescent substances, radioisotopes, enzymes that catalyze substrate color development, and chemiluminescent reagents.

[0205] In a preferred embodiment, the enzyme that catalyzes the color development of the substrate can be selected from, but not limited to, any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase.

[0206] In a preferred embodiment, the fluorescent substance can be selected from but not limited to any one of umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.

[0207] In a preferred embodiment, the chemiluminescent reagent may be lumefantrine, but is not limited thereto.

[0208] In a preferred embodiment, the radioisotope may be selected from but not limited to 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho and 153 Any of Sm.

[0209] In a sixth aspect, the present invention provides a method for diagnosing malaria, comprising the step of using the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect.

[0210] In a specific embodiment, the anti-malarial monoclonal antibody of the present invention can be an anti-HRP-II monoclonal antibody or an anti-LDH monoclonal antibody. The anti-malarial monoclonal antibody of the present invention or its antigen-binding fragment specifically binds to histidine-rich protein-II (HRP-II) or a fragment or variant thereof, and / or lactate dehydrogenase (LDH) or a fragment or variant thereof of the malaria parasite to detect malaria parasites, thereby diagnosing or detecting malaria.

[0211] In yet another specific embodiment, the malaria is caused by Plasmodium falciparum, a mutant Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale, such as Plasmodium falciparum and Plasmodium vivax.

[0212] As mentioned above, the antibody or antibody combination of the present invention can be used for pan-typing or typing detection of Plasmodium, thereby achieving universal detection or typing detection of malaria.

[0213] In a specific embodiment, the following antibody combination is used to detect malaria caused by Plasmodium falciparum, Plasmodium falciparum mutants, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale:

[0214] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 39-41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; or

[0215] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 49-51, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 52-54, respectively. L CDR1, V LCDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 71-73, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3.

[0216] In a further specific embodiment, the use of a combination of the above-mentioned anti-Plasmodium monoclonal antibodies or their antigen-binding fragments, such as a combination of recombinant antibodies L1' and L2' or a combination of L5' and L10', can diagnose malaria caused by Plasmodium falciparum, mutant Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale.

[0217] In yet another specific embodiment, the following antibody combination is used to detect Plasmodium vivax:

[0218] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 39-41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44, and SEQ ID NO: 41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0219] In a further specific embodiment, malaria caused by Plasmodium vivax can be diagnosed using a combination of anti-Plasmodium monoclonal antibodies or antigen-binding fragments thereof, such as a combination of recombinant antibodies L2' and L4'.

[0220] In yet another specific embodiment, the following antibody combination is used to detect Plasmodium falciparum or mutant Plasmodium falciparum:

[0221] The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequence of SEQ ID NO: 55-57, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, whose heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 86, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3.

[0222] In a further specific embodiment, malaria caused by Plasmodium falciparum or mutant Plasmodium falciparum can be diagnosed using a combination of anti-Plasmodium monoclonal antibodies or antigen-binding fragments thereof, such as a combination of recombinant antibodies L6' and L13'.

[0223] In an eighth aspect, the present invention provides use of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect in the preparation of a reagent for detecting Plasmodium and / or diagnosing malaria.

[0224] In a ninth aspect, the present invention provides a kit for detecting Plasmodium and / or diagnosing malaria infection, comprising the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the first aspect or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof of the second aspect; and instructions for use.

[0225] Again without wishing to be bound by theory, those skilled in the art will appreciate that the non-diagnostic detection methods of the present invention may include, but are not limited to, scientific research, quarantine testing, and the like.

[0226] The detection method of the present invention can be used in point-of-care testing (POCT) or electrochemical immunoassay systems. The detection method according to the present invention or any exemplary form thereof can be used in automated and semi-automated systems and optimized according to specific circumstances.

[0227] Example

[0228] The following examples illustrate the preparation methods and characterization of the relevant properties of the antibodies of the present invention. Unless otherwise specified, the experimental methods used are all conventional methods, and unless otherwise specified, the experimental materials used in the following examples are all purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0229] It should be noted that the terms used in the description of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The above summary of the invention, the detailed description, and the following examples are intended only to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the appended claims.

[0230] Example 1: Preparation of anti-malarial monoclonal antibodies

[0231] Immunization: The purified histidine-rich protein-Ⅱ (heavy chain biological, product number HP810-5) of Plasmodium falciparum was used as an immunogen to immunize mice. Specifically, female BALB / c mice aged 6-8 weeks were selected. The mice were immunized 4 times, with an interval of 2 weeks between each immunization, and the immunizing agent was 100 μg / mouse. For the first immunization, the histidine-rich protein-Ⅱ was mixed with Freund's complete adjuvant (Sigma-Aldrich) in equal volumes and injected subcutaneously at multiple points on the back. For the next three immunizations, the histidine-rich protein-Ⅱ was mixed with Freund's incomplete adjuvant (Sigma-Aldrich) in equal volumes and injected intraperitoneally. Seven days after the fourth immunization, the mice were bled by tail cutting, the serum was separated, and the antibody titer level of the antiserum of the immunized mice was detected by indirect ELISA to observe the immune response effect. Mice with serum antibody titers higher than 1:10000 were selected for cell fusion experiments. Three days before the cell fusion experiments, the mice were intraperitoneally injected with histidine-rich protein-II without adjuvant for booster immunization (100 μg / mouse).

[0232] Establishment of hybridoma cells: On the day of fusion, the spleen of the immunized mouse was removed under sterile conditions and the organ was made into a single cell suspension. Mouse myeloma cells (SP2 / 0) were fused with the above-mentioned immunized BALB / c mouse spleen cells at a ratio of 1:5, mixed thoroughly, and washed twice before fusion with PEG. Preheated PEG1500 was added, gently shaken, the cells were washed with preheated serum-free RPMI-1640 medium, and then resuspended with HAT selective medium. The cell suspension was plated into a 96-well culture plate at 200 μL / well and the cells were cultured at 37°C and 5% CO2. After culturing for 4 to 7 days, HT medium was used for culture. When the fused cells grew to 1 / 10-1 / 5 of the bottom area of ​​the wells of the 96-well plate, the supernatant was taken for antibody detection.

[0233] Screening of positive hybridoma cells: dilute histidine-rich protein-II with coating buffer (0.05 mol / L, pH 9.6, PBS) to a final concentration of 1 μg / mL, add 100 μL / well to a 96-well plate, and coat overnight at 4°C; discard the coating solution, wash three times with phosphate buffered saline (PBST), and pat dry; The plates were blocked with BSA in PBST (150 μL / well) and incubated at 37°C for 2 h, washed three times with PBST, and patted dry. The supernatant of the fused cells, 1:1000 diluted positive serum from immune mice (as a positive control), and 1:1000 diluted negative serum from mice (as a negative control) were added to the corresponding wells at 100 μL / well, incubated at 37°C for 1 h, washed three times with PBST, and patted dry. 100 μL / well of 1:4000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) was added and incubated at 37°C for 1 h, washed three times with PBST, and patted dry. 100 μL / well of tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) substrate was added and color was developed at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid was added to each well to terminate the reaction.

[0234] The OD of all wells was measured at 450 nm using a microplate reader. 450nm When the OD of negative serum 450nm When ≤0.1, the absorbance value OD of the measuring well (NS1) 450nm The value is the OD of the negative well 450nm The positive value was 2.1 times or more, and the positive hybridoma cells were selected for the next cloning step.

[0235] Cloning of positive cell lines: After sampling and counting the positive cell wells that secrete antibodies, dilute them to 100 cells / 10mL culture medium, plate the diluted cell suspension at 100μL / well to a 96-well cell culture plate, and culture in a 37°C, 5% CO2 cell culture incubator. After 6-7 days, the formation of cloned cells can be observed under a microscope. Mark the single clone growth well, remove the cell supernatant, perform ELISA test (the same as the above fusion test), and select positive monoclonal cells. Perform limiting dilution on the positive well cells, measure the ELISA value 5-6 days after each limiting dilution, and pick the OD value obtained by ELISA test. 450nm Limiting dilutions were performed on the wells containing the highest positive monoclonal counts until the entire 96-well plate was positive by ELISA. Stable monoclonal strains with high positive counts were selected. Finally, two cell lines that stably secreted anti-Plasmodium antibodies were obtained and designated hybridoma cell lines H1 and H2.

[0236] Preparation and purification of monoclonal antibodies on cells: Hybridoma cell lines were cultured in RPMI-1640 medium containing 15% serum in 10 cm culture dishes. When the culture was expanded to about 4×10 7 When the cells were 10 cells / dish, centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L spinner flask, add serum-free medium, and make the cell density about 3 × 10 5 After 1-2 weeks of culture, when the cell death rate reaches 80%-90% (the cell density is about 1×10 6 -2×10 6 The cell suspension was collected and centrifuged at 6000 rpm for 20 min. The supernatant was purified by Protein A immunochromatography.

[0237] The monoclonal antibodies produced by hybridoma cells were designated H1 and H2 antibodies. Microspectrophotometric analysis revealed concentrations of 3.57 mg / mL and 3.81 mg / mL, respectively, for H1 and H2. The purified monoclonal antibodies were diluted and aliquoted (100 μL / tube, 1 mg / mL concentration) and stored at 4°C-8°C until ready for use.

[0238] The two monoclonal antibodies were identified by SDS-PAGE electrophoresis. The antibodies had an antibody heavy chain band of about 51 KD and an antibody light chain band of about 26 KD.

[0239] Purity testing: Monoclonal antibodies were analyzed by size exclusion chromatography (SEC-HPLC). When all components in the sample were detected, the purity percentage of the main peak was calculated using the peak area normalization method. The purity was greater than 98%.

[0240] A similar method was performed using recombinant Plasmodium falciparum LDH protein (recombinant pf-LDH protein, heavy chain biological HP810-1) and recombinant Plasmodium vivax LDH protein (recombinant pv-LDH protein, heavy chain biological HP810-2) as immunogens, resulting in four cell lines (named hybridoma cell lines L1-L4, respectively) that secrete antibodies that specifically bind to pv-LDH (denoted as L1-L4 antibodies) and ten cell lines (named hybridoma cell lines L5-L14, respectively) that secrete antibodies that specifically bind to pf-LDH (denoted as L5-L16 antibodies).

[0241] Example 2: Antibody-antigen binding ability

[0242] Histidine-rich protein-II, recombinant pf-LDH, and pv-LDH proteins were diluted in 0.05 M carbonate buffer (pH 9.6) to a concentration of 1 μg / mL. 100 μL / well was added to a 96-well microtiter plate and coated overnight at 4°C. The plates were washed three times with PBST on an automated plate washer and patted dry. Blocking was performed with PBST containing 2% BSA (150 μL / well), incubated at 37°C for 2 h, washed three times with PBST, and patted dry. Anti-Plasmodium monoclonal antibodies were serially diluted in 0.02 M PBS buffer (pH 7.4) starting at a concentration of 5 μg / mL. Three-fold serial dilutions were then performed to generate a series of monoclonal antibody samples at varying concentrations. The diluted monoclonal antibody samples were added to the plate at 100 μL / well, incubated at 37°C for 1 h, washed three times, and patted dry. Add 100 μL / well of a 1:4000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma) and incubate at 37°C for 1 hour. Wash three times with PBST and pat dry. Add 100 μL / well of TMB substrate and develop for 10 minutes at room temperature in the dark. Terminate the reaction by adding 50 μL / well of 2 mol / L sulfuric acid.

[0243] OD was measured using a microplate reader 450nm The EC value of the monoclonal antibody binding to the antigen was obtained by analyzing the above ELISA results through software. 50 Value (nM).

[0244] Figure 1 shows the binding reaction results of monoclonal antibodies H1 and H2 with histidine-rich protein-II and Plasmodium falciparum pf-LDH protein (as a control). As can be seen from Figure 1, the screened monoclonal antibodies H1 and H2 can both specifically bind to histidine-rich protein-II but not to pf-LDH protein, demonstrating good specificity.

[0245] In addition, Table 1 shows the EC values ​​of each monoclonal antibody and the corresponding antigen. 50 The values ​​are as follows:

[0246] Table 1: EC of antibody-antigen binding 50 Value (nM)

[0247] Example 3: Cloning and sequencing of antibody variable region sequences

[0248] Total RNA was isolated from the hybridoma cell line, and cDNA was prepared by reverse transcription. Immunoglobulin sequences were cloned from the hybridoma cell line, and the variable region sequences of the antibodies of the hybridoma cell line were determined.

[0249] a. RNA extraction: Referring to the instructions of the M5 total RNA extraction kit (purchased from Beijing PolyBiotech Co., Ltd.), total RNA was extracted from the above hybridoma cell lines and immediately reverse transcribed.

[0250] b. RNA was reverse transcribed into cDNA: The total RNA extracted in the previous step was reverse transcribed using the M5 First Strand cDNA Synthesis Kit (purchased from Beijing Polymer Biotechnology Co., Ltd.) to produce cDNA, which was then frozen at -20°C for later use.

[0251] c. PCR amplification and recovery of variable region sequences: The cDNA obtained in the previous step was used as a template and the universal heavy chain primer Mu Ig V H 5′-A and Mu IgG V H 3′-2, immunoglobulin heavy chain (IgH) cDNA was amplified by PCR; similarly, light chain primer Mu IgκV L 5′-A and Mu IgκV L 3′-1, immunoglobulin light chain (IgK) cDNA was amplified by PCR. The PCR product was then recovered; the entire PCR reaction used thermostable Pfu DNA polymerase.

[0252] d. Cloning and sequencing of variable region sequences: According to the instructions of the cloning vector pTOPO-Blunt Cloning kit (purchased from Beijing Polymer Biotechnology Co., Ltd.), the heavy and light chain variable region genes were ligated to the pTOPO vector, transformed into Escherichia coli DH5α, and positive clones were selected and sequenced by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0253] The antibody heavy chain variable region gene sequences and light chain variable region gene sequences of the hybridoma cell lines obtained by sequencing were analyzed. The heavy chain complementary determining region sequences and light chain complementary determining region sequences are shown in Table 2 below (based on the Chothia numbering system).

[0254] Table 2: Complementarity determining region sequences of heavy and light chains of antibodies

[0255] Example 4: Preparation and purification of recombinant antibodies

[0256] Through genetic recombination technology, point mutations were performed on some sites in the complementary determining regions of the heavy and light chains mentioned above, and a series of recombinant antibodies with point mutations were constructed. Cell lines stably expressing antibodies were prepared through eukaryotic expression, and then cultured and purified on a large scale.

[0257] For V encoding the anti-LDH monoclonal antibodies listed in Table 2 aboveL CDR and V H The CDR genes and the V genes of the anti-HRP-II monoclonal antibodies listed in Table 3 below L CDR and V H The CDR gene, combined with the V shown in SEQ ID NO: 25-28 H FR1-V H FR4 and V shown in SEQ ID NO: 29-32 L FR1-V L FR4 (for antibody H1'-H20'), V shown in SEQ ID NO: 97-100 H FR1-V H FR4 and V shown in SEQ ID NO: 101-104 L FR1-V L FR4 (for antibody L1'-L4') and V shown in SEQ ID NO: 89-92 H FR1-V H FR4 and V shown in SEQ ID NO:93-96 L FR1-V L FR4 (for antibody L5'-L14'), a eukaryotic expression plasmid for the recombinant antibody was constructed using molecular cloning methods. This eukaryotic expression plasmid was electroporated into CHO host cells and cultured in pressure selection medium (50 μM MSX) for 20 days. The supernatant was then analyzed by ELISA (using HRP-labeled goat anti-mouse IgG as a secondary antibody, using the same method as above) to identify cell lines stably expressing the recombinant antibody. The specific sequence correspondences of the heavy and light chain complementary determining regions of the recombinant antibody are shown in Table 3 below.

[0258] Table 3: Sequences of the heavy and light chain complementary determining regions of the recombinant anti-HRP-II antibody H1'-H20'

[0259] The selected stable cell lines were cultured on a large scale using roller bottle culture technology for the preparation of recombinant antibodies. The cells were cultured in Vega CHO medium at a rate of (0.2-0.3) × 10 6Cells / ml were inoculated into roller bottles. A 1L roller bottle contained 300ml of culture medium (Vega CHO). The number of inoculated bottles was determined based on production needs. The roller bottles inoculated with cells were placed in a cell roller machine and cultured in a cell culture incubator. The culture conditions were 900 rpm, 37°C, and 5% carbon dioxide. After 7-9 days of culture, samples were taken for observation under a microscope. When the cell viability was less than 50%, the samples were collected by centrifugation. The samples were affinity purified using a protein A affinity chromatography column to obtain the antibodies, namely the recombinant anti-HRP-Ⅱ monoclonal antibodies H1'-H20' and the recombinant anti-LDH monoclonal antibodies L1'-L14'.

[0260] The binding ability of recombinant antibodies H1'-H20' to HRP-II was tested using the method described in Example 2. The binding data for recombinant antibodies H1'-H15' to HRP-II and pf-LDH protein (as a control) are shown in Figure 2. As shown in Figure 2, recombinant antibodies H1'-H15' bind strongly to HRP-II, demonstrating good affinity, while not binding to pf-LDH protein, indicating good specificity. Other recombinant antibodies H16'-H20' (results not shown) also exhibited good affinity and specificity for HRP-II.

[0261] Furthermore, the obtained OD 450nm The values ​​were analyzed by software to obtain the EC values ​​of each recombinant antibody. 50 The values ​​are shown in Table 4 below.

[0262] Table 4: EC of the recombinant monoclonal antibodies of the present invention and the corresponding antigens 50 Value (nM)

[0263] From the results given in Table 4, it can be seen that all the recombinant monoclonal antibodies have the same EC as HRP-Ⅱ. 50 All of them were in the nM level, and the recombinant H8', H13' and H16' showed the best EC 50 In addition, it can be seen from the results in Table 4 that the binding ability of the recombinant anti-LDH monoclonal antibody to pf-LDH / pv-LDH is basically consistent with that of its corresponding monoclonal antibody.

[0264] Example 5: Colloidal gold immunochromatographic test

[0265] Preparation of colloidal gold: Add 200 mL of ultrapure water to a conical flask and heat to boiling. Add 1 mL of 2% chloroauric acid (Sigma-Aldrich, Catalog No. 16961-25-4) solution. Immediately after boiling, add 1 mL of 2% trisodium citrate (Sigma-Aldrich, Catalog No. 6132-04-3) aqueous solution. Continue stirring and boiling for 10 minutes. Cool naturally and set aside.

[0266] Labeling colloidal gold conjugate: Take 10mL of the above colloidal gold and place it in a beaker. Add 120μL of 0.2M K2CO3 to adjust the pH to 7.0 while stirring, and continue stirring for 10 seconds; add 100μg of labeled antibody (antibody A) and continue stirring for 5 minutes; add 0.1mL of 10% BSA and continue stirring for 5 minutes; centrifuge at 12000g for 10 minutes, discard the supernatant, and dilute the precipitate to 1mL with colloidal gold diluent (10mM PB, 150mM NaCl, 0.2% BSA, 0.1% TritonX-100, 3% Sucrose, 0.01% Proclin300) to prepare the anti-HRP-Ⅱ / LDH antibody colloidal gold complex.

[0267] Preparation of colloidal gold pads: The colloidal gold complexes were diluted 10 times with colloidal gold diluent, soaked in glass fiber (Shanghai Jinbiao Company), and freeze-dried to prepare gold label pads.

[0268] Nitrocellulose membrane (NC membrane) coating: Dilute the coating antibody (Antibody B) to 1 mg / mL to prepare the detection line working solution. Use a membrane spotter to draw lines on the corresponding positions of nitrocellulose membrane (Millipore, Cat. No.: HF135002). Dry at 50°C for 1 hour and set aside.

[0269] Assembly of colloidal gold immunochromatographic test reagent strip: Assemble the above-mentioned gold label pad, nitrocellulose membrane coated with antibody, absorbent paper, polyester plate, and sample pad into a colloidal gold immunoassay reagent strip.

[0270] Sensitivity test: Samples with different concentrations of recombinant antigens were tested separately. Specifically, 80 μL of the sample to be tested was added to the sample pad, left at room temperature for 5-15 minutes, and the result was determined. The activity of the antigen-antibody binding in the sample can be indicated by the depth of the displayed strip color. Compare the color of the T-line strip developed by the colloidal gold test paper with the standard color card, select the closest color, and mark the activity of the product with the color number corresponding to the color. Use pf-LDH protein (for HRP-II), HRP-II (for pv-LDH) and HRP-II (for pf-LDH) as negative controls, respectively. The results are shown in Table 5 below, where the coating antibody concentration is 1 mg / mL, and 10 μg of labeled antibody is coupled to each 1 mL of colloidal gold; B represents blank, i.e., no binding; + represents half a level, for example, C5+ is a strip whose color is between C5 and C4.

[0271] Table 5: Colloidal gold immunochromatographic test results of the antibody combination of the present invention

[0272] The above results show that the minimum detection limit of the colloidal gold chromatography reagent made from the anti-HRP-Ⅱ antibody or anti-pv-LDH antibody of the present invention for recombinant antigens can reach 0.5 ng / mL, and the minimum detection limit of the colloidal gold chromatography reagents of some antibody pairs can even reach 0.1 ng / mL, while the minimum detection limit of the colloidal gold chromatography reagent made from the anti-pf-LDH antibody of the present invention for recombinant antigens can reach 1 ng / mL (using color card grade 8 as the positive standard).

[0273] In addition, the inventors also tested the sensitivity of all antibody combinations using the same method, and the results are shown in Tables 6-7.

[0274] Table 6: Sensitivity results of colloidal gold immunochromatographic test using anti-pv-LDH monoclonal antibody combination

[0275] Table 7: Sensitivity results of colloidal gold immunochromatographic test using anti-pf-LDH monoclonal antibody combination

[0276] Note: "High" refers to color card grades 1-2, "higher" refers to 3-4, "lower" refers to 5-6, and "low" refers to 7-9.

[0277] In addition, the inventors further tested three serum samples from patients with falciparum malaria, namely strong positive, moderate positive and weak positive, using the product of manufacturer A as a control. The comparison results are shown in Table 8 below.

[0278] Table 8: Colloidal gold immunochromatographic test results of the anti-HRP-II antibody combination of the present invention

[0279] Furthermore, the inventors tested strongly positive, moderately positive (two) and weakly positive serum samples from patients with Plasmodium vivax malaria using the product of manufacturer B as a control. The comparison results are shown in Table 9 below.

[0280] Table 9: Colloidal gold immunochromatographic test results of the anti-pv-LDH antibody combination of the present invention

[0281] Furthermore, the inventors tested strongly positive, moderately positive (two) and weakly positive serum samples from patients with falciparum malaria using the product of manufacturer C as a control. The comparison results are shown in Table 10 below.

[0282] Table 10: Colloidal gold immunochromatographic test results of the anti-pf-LDH antibody combination of the present invention

[0283] The results showed that the sensitivity of the colloidal gold chromatography reagent composed of the anti-HRP-Ⅱ monoclonal antibody of the present invention was comparable to that of the product of manufacturer A, and the detection sensitivity of the colloidal gold chromatography reagent of partial pairing (such as H4' as the coating antibody and H15' as the labeling antibody) for weakly positive serum was better than that of the product of manufacturer A; the sensitivity of the colloidal gold chromatography reagent composed of the anti-pv-LDH monoclonal antibody of the present invention was comparable to that of the product of manufacturer B, and the detection sensitivity of the colloidal gold chromatography reagent of partial pairing (such as L1' as the coating antibody and L4' as the labeling antibody; and L2' as the coating antibody and L4' as the labeling antibody) for weakly positive, moderately positive and strongly positive serum was better than that of the product of manufacturer B; the sensitivity of the colloidal gold chromatography reagent composed of the anti-pf-LDH monoclonal antibody of the present invention was slightly better than that of the product of manufacturer C. For example, for the moderately positive sample 1, the anti-pf-LDH monoclonal antibody combination of the present invention showed higher detection sensitivity.

[0284] Specificity testing: Using the HRP-II colloidal gold chromatography reagent from manufacturer A as a control, a reagent strip prepared with the anti-HRP-II antibody of the present invention was tested on 300 serum samples from healthy individuals. The results showed that the reagent strip prepared with the anti-HRP-II antibody of the present invention had no false positive results, with a specificity of 100%. Similarly, 100 serum samples each from healthy individuals were tested using reagent strips prepared with the anti-pf-LDH antibody and anti-pv-LDH antibody of the present invention. The results showed that the reagent strips prepared with the anti-pf-LDH antibody and anti-pv-LDH antibody of the present invention had no false positive results, with a specificity of 100%.

[0285] Example 6: Malaria typing

[0286] Recombinant monoclonal antibodies L1'-L4' and L5'-L14' were used to prepare a colloidal gold immunoassay reagent strip according to the method in Example 5. The reagent strip was used to detect pf-LDH, pv-LDH, po-LDH, pm-LDH and HRP-Ⅱ positive samples (sera from positive patients). The test results are shown in Table 11 below.

[0287] Table 11: Results of colloidal gold immunochromatographic tests of the recombinant LDH antibody combination of the present invention

[0288] The test results show that the combination of L4' as the coating antibody and L2' as the labeling antibody can detect pv-LDH serum samples, while the reaction results with po-LDH, pm-LDH, pf-LDH and HRP-Ⅱ serum samples are negative; the combination of L2' as the coating antibody and L1' as the labeling antibody and the combination of L10' as the coating antibody and L5' as the labeling antibody can detect four positive sera such as po-LDH, pm-LDH, pv-LDH, pf-LDH and HRP-Ⅱ positive serum; the combination of L6' as the coating antibody and L13' as the labeling antibody can detect pf-LDH and HRP-Ⅱ positive sera, while the reaction with po-LDH, pm-LDH and pv-LDH sera is negative.

[0289] The combination of H1' as a coating antibody and H5' as a labeled antibody can detect pf-LDH serum samples and HRP-II serum samples. It is worth noting that the term "detection" here does not mean that the anti-HRP-II antibodies H1' or H5' of the present invention can bind to the pf-LDH protein. Rather, because the pf-LDH and HRP-II positive serum samples are all from patients with falciparum malaria, such serum samples must contain both pf-LDH and HRP-II proteins and are therefore detectable by the combination of antibodies H1' and H5'. Distinguishing these positive sera as pf-LDH and HRP-II is based on different classification indicators and does not mean that only pf-LDH or HRP-II proteins are involved in such sera.

[0290] Furthermore, the above L2'+L1' antibody combination and L10'+L5' antibody combination were used to detect different concentrations of recombinant antigens pv-LDH, pf-LDH, pm-LDH, po-LDH and positive cultures, and the results are shown in Table 12 below.

[0291] Table 12: Colloidal gold immunochromatographic test results of the recombinant antibody combination of the present invention

[0292] As can be seen from the results in Table 12 above, the colloidal gold results of the exemplary two-antibody combination showed that the recombinant LDH or culture of four types of malaria could be detected.

Claims

1. An anti-malarial monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to histidine-rich protein-II (HRP-II) of malaria parasite or a fragment or variant thereof, or specifically binds to lactate dehydrogenase (LDH) of malaria parasite or a fragment or variant thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementary determining region V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementarity determining region V L CDR1, V L CDR2 and V L CDR3; In an antibody or antigen-binding fragment thereof that specifically binds lactate dehydrogenase (LDH) or a fragment or variant thereof: V H The amino acid sequence of CDR1 is represented by X1-X2-X3-X4-X5-X6-X7, wherein X1 is G or E, X2 is Y or F, X3 is T or S, X4 is F or L, X5 is T, F or S, X6 is S, T or N, and X7 is S or Y; V H The amino acid sequence of CDR2 is represented by X8-X9-X10-X11-X12-X13, wherein X8 is D, N, W or Y, X9 is P, S or A, X10 is S, Y or G, X11 is absent or is D, S or N, X12 is S, D or G, and X13 is F, G, D, T, N or Y; V H The amino acid sequence of CDR3 is represented by X14-X15-G-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-Y, wherein X14 is absent or GN, X15 is absent, L or Y, X16 is S, N, D, X17 is S, N, I, X18 is absent or Y, X19 is D, L or G, X20 is E, R or Y, X21 is D, E, G or N, X22 is F, Y, T, N or G, X23 is GN, N, V, S, G, Y or H, X24 is A, T, G or S, X25 is M, L, F or I, and X26 is D or T; V L The amino acid sequence of CDR1 is represented by X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39-X40, wherein X27 is RAS or absent, X28 is S, T, Q or K, X29 is A, V or S, X30 is S or V, X31 is A, S, E or Q, X32 is S, N or T, X33 is I, V, F or L or absent, X34 is S, T, G or L, X35 is S, Y, T or N, X36 is S, R, Y or T, X37 is R, T, K, N or absent, X38 is Y, V or S, X39 is S, L or M, and X40 is H, A or absent; V L The amino acid sequence of CDR2 is represented by X41-X42-S-X43-X44-X45-X46, wherein X41 is S, G, W or F, X42 is T or A, X43 is N or T, X44 is L or R, X45 is A, Y, C or E, and X46 is S, T or I; V L The amino acid sequence of CDR3 is represented by X47-X48-X49-X50-X51-X52-X53-X54-T, wherein X47 is H, G, Q or K, X48 is Q or H, X49 is Y, S or T, X50 is H, F, W or Y, X51 is R, H, N or E, X52 is S, Y, L or I, X53 is P or absent, and X54 is W, P or Y; In an antibody or antigen-binding fragment thereof that specifically binds to histidine-rich protein-II or a fragment or variant thereof: V H The amino acid sequence of CDR1 is shown by SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 14 or SEQ ID NO: 19, or by a sequence having 80% or more, 85% or more, 90% or more identity to said sequence; V H The amino acid sequence of CDR2 is shown by SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:15 or SEQ ID NO:20, or by a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity to said sequence; V H The amino acid sequence of CDR3 is shown by SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:21, or by a sequence having 80% or more, 85% or more, 90% or more identity to said sequence; V L The amino acid sequence of CDR1 is shown by SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:17 or SEQ ID NO:22, or by a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity to said sequence; V L The amino acid sequence of CDR2 is shown by SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:23, or by a sequence having 80% or more, 85% or more, 90% or more identity to said sequence; and V L The amino acid sequence of CDR3 is shown by SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18 or SEQ ID NO:24, or by a sequence having more than 80%, more than 85%, more than 90% or more identity with the sequence.

2. The anti-malarial monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: 1) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 33-35, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 36-38 respectively. L CDR1, V L CDR2 and V L CDR3; 2) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 39-41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO:42, SEQ ID NO:37 and SEQ ID NO:43, respectively. L CDR1, V L CDR2 and V L CDR3; 3) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44 and SEQ ID NO: 41, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; 4) the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively; H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; 5) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 49-51, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 52-54 respectively. L CDR1, V L CDR2 and V L CDR3; 6) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 55-57, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L CDR3; 7) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 60-62, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 53 and SEQ ID NO: 64, respectively. L CDR1, V L CDR2 and V L CDR3; 8) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 56 and SEQ ID NO: 65, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 53 and SEQ ID NO: 67, respectively. L CDR1, V L CDR2 and V L CDR3; 9) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 68, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 69 and SEQ ID NO: 70, respectively. L CDR1, V L CDR2 and V L CDR3; 10) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 71-73, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3; 11) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 77 and SEQ ID NO: 73, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 78, SEQ ID NO: 75 and SEQ ID NO: 79, respectively. L CDR1, V L CDR2 and V L CDR3; 12) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 49, SEQ ID NO: 80 and SEQ ID NO: 81, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 82-84 respectively. L CDR1, V L CDR2 and V L CDR3; 13) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 86, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3; 14) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 88, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and VL CDR3; 15) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively. L CDR1, V L CDR2 and V L CDR3; 16) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 7-9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 10-12 respectively. L CDR1, V L CDR2 and V L CDR3; 17) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; 18) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3; 19) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 13, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3; 20) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 14-16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 22-24 respectively. L CDR1, V L CDR2 and V L CDR3; 21) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively. L CDR1, V L CDR2 and V L CDR3; 22) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 23) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 2 and SEQ ID NO: 16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; 24) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3; 25) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; 26) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 27) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 13, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises The amino acid sequences of the light chain complementary determining regions V and V are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; 28) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 14-16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3; 29) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 16, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 30) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 2 and SEQ ID NO: 21, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; 31) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 21, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 24, respectively. L CDR1, V L CDR2 and V L CDR3; 32) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 20 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 18, respectively. L CDR1, V L CDR2 and V L CDR3; 33) The heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 8 and SEQ ID NO: 3, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. L CDR1, V L CDR2 and V L CDR3; or 34) The heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 19-21, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 22-24 respectively. L CDR1, V L CDR2 and V L CDR3.

3. The anti-malarial monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, and the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4, wherein: 1) The heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown in SEQ ID NOs: 25-28, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences; the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown in SEQ ID NOs: 29-32, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences; 2) the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown in SEQ ID NOs: 89-92, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences; the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown in SEQ ID NOs: 93-96, or sequences having 80% or more, 85% or more, 90% or more identity with the sequences; 3) The heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 respectively have the sequences shown by SEQ ID NOs:97-100, or sequences having more than 80%, more than 85%, more than 90% or more than 95% identity with the sequences; the light chain framework regions LFR1, LFR2, LFR3 and LFR4 respectively have the sequences shown by SEQ ID NOs:101-104, or sequences having more than 80%, more than 85%, more than 90% or more than 95% identity with the sequences.

4. The anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the lactate dehydrogenase (LDH) is one or more of pf-LDH, pv-LDH, pm-LDH and po-LDH.

5. A combination of anti-malarial monoclonal antibodies or antigen-binding fragments thereof, comprising: 1) a first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of the antibodies 1) to 4) described in claim 2; Preferably, the first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region comprising a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L The second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region including a heavy chain complementary determining region V represented by amino acid sequences of SEQ ID NOs: 39-41, H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3 antibody or antigen-binding fragment thereof; or The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region including heavy chain complementary determining regions V and V represented by amino acid sequences of SEQ ID NOs: 39-41, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L The second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region including a heavy chain complementary determining region V represented by amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44 and SEQ ID NO: 41, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L An antibody or antigen-binding fragment thereof targeting CDR3; 2) a first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of antibodies 5) to 14) described in claim 2; Preferably, the first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region comprising a heavy chain complementary determining region V having an amino acid sequence represented by SEQ ID NOs: 49-51, H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 and the light chain variable ... L CDR1, V L CDR2 and V L CDR3 antibody or antigen-binding fragment thereof, the second anti-Plasmodium monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region includes the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 71-73, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 and the light chain variable ... L CDR1, V L CDR2 and V L CDR3 antibody or antigen-binding fragment thereof; or The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof has a heavy chain variable region including heavy chain complementary determining regions V and V represented by amino acid sequences of SEQ ID NOs: 55-57, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L The second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region including a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 86, respectively. H CDR1, V H CDR2 and V H and a light chain variable region comprising a light chain complementary determining region V having amino acid sequences represented by SEQ ID NO: 87, SEQ ID NO: 83, and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L or an antibody or antigen-binding fragment thereof that is directed against CDR3; or 3) A first anti-Plasmodium monoclonal antibody and a second anti-Plasmodium monoclonal antibody independently selected from any one of the antibodies 15) to 38) described in claim 2.

6. A nucleic acid molecule encoding the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. A vector comprising the nucleic acid molecule according to claim 6 .

8. The carrier according to claim 7, wherein The vector is a plasmid vector.

9. The carrier according to claim 7, wherein The plasmid vector is any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

10. An expression cell comprising the vector according to any one of claims 7 to 9.

11. The expression cell according to claim 10, wherein The expression cells are mammalian cells.

12. The expression cell according to claim 11, wherein The mammalian cells are Chinese hamster ovary cells, miniature hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

13. A method for detecting Plasmodium, comprising the step of using the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to claim 5.

14. The method according to claim 13, wherein: The malarial parasite includes any one or more of Plasmodium falciparum, mutant Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale; For example, the following antibody combination was used to detect all of Plasmodium falciparum, Plasmodium falciparum mutants, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale: The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: Heavy chain complementary determining region V shown in 39-41 H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO:42, SEQ ID NO:37 and SEQ ID NO:43, respectively. L CDR1, V L CDR2 and V L CDR3; or The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 49-51, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 52-54, L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 71-73, respectively; H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 and the light chain variable region V CDR4, which are represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3; or As another example, the following antibody combination is used to detect Plasmodium vivax: The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 39-41, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44 and SEQ ID NO: 41, respectively; H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; or Detection of P. falciparum or mutant P. falciparum using the following antibody combinations: The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 55-57, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 86, respectively; H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3.

15. The method according to claim 13 or 14, wherein: The detection is carried out by immunochromatography, enzyme-labeled antibody method (ELISA), chemiluminescence method, electrochemiluminescence method; preferably, the immunochromatography includes fluorescent microsphere immunochromatography, colloidal gold immunochromatography, immunochromatography based on colored latex microspheres, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography and quantum dot immunochromatography; ELISA such as direct method, indirect method, sandwich method and competitive method.

16. A method for diagnosing malaria, comprising the step of using the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or a combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to claim 5.

17. The method according to claim 16, wherein: The malaria is caused by Plasmodium falciparum, Plasmodium falciparum mutant, Plasmodium vivax, Plasmodium malariae or Plasmodium ovale; For example, use the following antibody panel to detect malaria caused by Plasmodium falciparum, Plasmodium falciparum mutants, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale: The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 46 and SEQ ID NO: 47, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 39-41, respectively; H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO:42, SEQ ID NO:37 and SEQ ID NO:43, respectively. L CDR1, V L CDR2 and V L CDR3; or The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 49-51, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 52-54, L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 71-73, respectively; H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V CDR3 and the light chain variable region V CDR4, which are represented by the amino acid sequences of SEQ ID NOs: 74-76, respectively. L CDR1, V L CDR2 and V L CDR3; or As another example, the following antibody combination is used to detect malaria caused by Plasmodium vivax: The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 39-41, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 44 and SEQ ID NO: 41, respectively; H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 37 and SEQ ID NO: 43, respectively. L CDR1, V L CDR2 and V L CDR3; or Detect malaria caused by Plasmodium falciparum or mutant Plasmodium falciparum using the following antibody combination: The first anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 55-57, respectively. H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 59, respectively. L CDR1, V L CDR2 and V L CDR3; a second anti-Plasmodium monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 85, SEQ ID NO: 56 and SEQ ID NO: 86, respectively; H CDR1, V H CDR2 and V H The light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 87, SEQ ID NO: 83 and SEQ ID NO: 84, respectively. L CDR1, V L CDR2 and V L CDR3.

18. Use of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the combination of the anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to claim 5 in the preparation of a reagent for detecting Plasmodium and / or diagnosing malaria.

19. A kit for detecting Plasmodium and / or diagnosing malaria infection, comprising: An anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a combination of an anti-Plasmodium monoclonal antibody or antigen-binding fragment thereof according to claim 5; and instructions for use.

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