CN111378035A

CN111378035A - Anti-plasmodium falciparum HRP-II recombinant antibody

Info

Publication number: CN111378035A
Application number: CN201811654711.5A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 唐丽娜; 梁碧; 游辉
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2020-07-07
Anticipated expiration: 2038-12-29
Also published as: CN111378035B

Abstract

The invention relates to an anti-plasmodium falciparum HRP-II recombinant antibody. The present invention provides an anti-plasmodium falciparum HRP-II antibody, or antigen-binding fragment thereof, comprising SEQ ID NO: 1-3 or a variant thereof, SEQ ID NO: 4-6 or a variant thereof. The invention provides uses of the antibodies and methods of making the antibodies.

Description

Anti-plasmodium falciparum HRP-II recombinant antibody

Technical Field

The present invention relates to plasmodium antibodies, their use and methods of preparation. In particular, the present invention relates to antibodies to Histidine-rich protein II (HRP-II) of Plasmodium falciparum, their use for the detection or diagnosis of malaria, and methods of making the antibodies by genetic engineering techniques.

Background

Malaria (Malaria) is an insect-borne infectious disease caused by infection of plasmodium by the bite of the anopheles mosquito or transfusion into the blood of a person with the plasmodium. There are four main species of plasmodium parasitic to humans: plasmodium vivax (pv), Plasmodium falciparum (Pf), Plasmodium malaciparum (pm), Plasmodium ovale (po). Plasmodium falciparum is a common infectious plasmodium (75%) and the most harmful pathogen, has strong infectivity, rapid proliferation, severe symptoms and high lethality rate of primary infected persons, causes more than 95% of death of infected persons, and is mainly present in tropical regions of africa, south america and asia; plasmodium vivax is the second most common plasmodium (20%) and is also the most common species in areas other than continent. WHO recommends that all suspected malaria patients should be tested for malaria in real time, and the rapid and accurate diagnosis of the disease is crucial to the correct use of antimalarial drugs, the prevention of drug-resistant strains, the control of disease deterioration, and the reduction of mortality.

Malaria diagnosis is the focus of malaria control efforts. The current methods for detecting plasmodium can be classified into four categories, based on the principle of detection technology. One is the microscope to directly detect plasmodium, including thick blood membrane and thin blood membrane, which is also the current gold standard for clinical malaria diagnosis. But is time consuming, labor intensive, and requires skilled personnel and certain experimental conditions. Secondly, plasmodium nucleic acid detection, in which a detection target is specific nucleotide fragments such as plasmodium 18S ribosomal RNA and the like, and common methods are a fluorescence PCR method and a loop-mediated isothermal amplification (LAMP). Although the method has high sensitivity and specificity, the method needs more complex instruments and technical conditions as support, is not suitable for being used as a conventional detection means in malaria epidemic areas, and is difficult to popularize and apply at the basic level. And thirdly, detecting hemozoin by adopting a flow cytometry detection technology or a mass spectrometry method. The method needs a professional detection instrument, is generally used for laboratory research and is not suitable for field detection. Fourthly, the immune reaction of the antigen and the antibody detects the plasmodium, the methodology comprises an immunochromatography rapid diagnostic Reagent (RDT) and enzyme-linked immunosorbent assay (ELISA), and most of the detected target antigens are diagnostic antigens such as LDH, HRP-II and the like. The RDT taking the antigen as the detection target has important significance in the laggard areas of the prevalence of plasmodium falciparum, and is a method recommended by WHO for on-site diagnosis due to the advantages of simple and convenient operation, quickness, intuitive result, no need of complex equipment, high sensitivity and specificity and the like.

Currently, the commonly used detection malaria antigens are mainly Histine-rich protein II (HRP-II) and Plasmodium Lactate Dehydrogenase (PLDH) specific to Plasmodium falciparum. HRP-II is a 35-105kDa water-soluble protein specific to Plasmodium falciparum, which is synthesized only in the anaplasmodium falciparum aneuploidy stage and the early gametophyte stage, starting from immature circular bodies, and the content gradually increases along with the fission of merozoites in the whole erythrocytic stage, can last for 28 days, and is difficult to detect in the mature gametophyte stage. The HRP-II protein is secreted into the blood of a host in a soluble form, and can be detected in cerebrospinal fluid, urine and saliva, so that the HRP-II protein is the most accepted diagnostic marker for plasmodium falciparum. The presence of Plasmodium falciparum in humans can be judged by detecting HRP-II with a specific monoclonal antibody.

At present, many kits for detecting plasmodium by using HRP-II as a target protein are available in the market, such as a foreign Parasight-F kit, ICT Malaria P.f.&P.f/P.v A diagnostic kit,

a kit, a CareStart malaria HRP-II/PLDH composite test kit; there are Aikang P.f/P.v colloidal gold immunochromatography detection kits and the like in China. The Parasight-F kit is an application product recommended by the world health organization, a mouse anti-HRP-II IgG1 monoclonal antibody and a control HRP-II antigen are coated on a strip-shaped nitrocellulose membrane in a linear and dotted line shape to be respectively used as a capture antibody and a detection line, and sulforhodamine B is used as an indicator for developing color, so that only malignant malaria can be detected. An ICT Malaria P.f/P.v diagnostic kit coats 1 strain of anti-plasmodium falciparum HRP-II monoclonal antibody and 1 strain of plasmodium vivax species specific monoclonal antibody on a nitrocellulose membrane as capture antibodiesIt is used for developing color by colloidal gold label, and is used for malignant malaria, vivax malaria and mixed infection.

The kit applies 2 different monoclonal antibodies, the detection target protein is HRP-II and aldolase, the HRP-II is peculiar to plasmodium falciparum, the aldolase is a common component of 4 plasmodium falciparum infecting human body, and meanwhile, the kit detects simple plasmodium falciparum and other plasmodium falciparum except the plasmodium falciparum. The CareStart malaria HRP-II/PLDH composite test kit forms two independent detection lines on a membrane by using a single clone monomer in 2, wherein the two detection lines are respectively a lactate dehydrogenase (PLDH) monoclonal antibody and an anti-HRP-II monoclonal antibody of anti-plasmodium (plasmodium falciparum, vivax, oval malaria and triumnial malaria) and are specially used for differential diagnosis of the plasmodium falciparum and other types of malaria. Aikang P.f/P.v colloidal gold immunochromatography detection kit respectively adopts anti-plasmodium falciparum HRP-II and anti-plasmodium vivax PLDH monoclonal antibodies as capture antibodies, and simultaneously diagnoses infection of plasmodium falciparum and plasmodium vivax.

In each of the above kits, an anti-HRP-II monoclonal antibody was used. At present, the conventional preparation method of monoclonal antibodies for diagnosis in the market is a hybridoma technology, namely, a gene engineering technology is utilized to express HRP-II protein to immunize mice, spleen cells and tumor cells of the immunized mice are fused to obtain hybridoma cells, and finally, the hybridoma cells secreting target antibodies are screened out and then antibody production is carried out.

Disclosure of Invention

The invention provides anti-plasmodium falciparum HRP-II antibodies or antigen-binding fragments thereof. The invention also provides a preparation method of the antibody, an antibody conjugate, a fusion protein and a kit/diagnostic agent containing the antibody, and application of the antibody in diagnosing plasmodium falciparum infection.

In some embodiments, the invention provides an antibody, or antigen-binding fragment thereof, that binds plasmodium falciparum HRP-II, wherein the antibody comprises the following amino acid sequence or complementarity determining regions having at least 80% sequence identity thereto (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity):

a heavy chain CDRl comprising SEQ ID NO: 1, or consists of, the amino acid sequence G-Y-X1-F-X2-S-Y-W-X3-H, wherein X1 is S, Y or T; x2 is S or T, preferably T; x3 is L, V or I;

a heavy chain CDR2 comprising SEQ ID NO: 2, or consists of, wherein Xl is a or P, preferably P; x2 is R or K; x3 is N, R or Q; and

a heavy chain CDR3 comprising SEQ ID NO: 3, or consists of, wherein Xl is I or L, preferably I; x2 is Y or F; x3 is Q, R or N;

and the antibody further comprises:

a light chain CDR1 comprising SEQ ID NO: 4, or consists of, an amino acid sequence S-K-S-X1-X2-Q-T-N-X3-N-X4-Y-L-Y, wherein X1 is I or L; x2 is L or I; x3 is P or G, preferably G; x4 is S or T;

a light chain CDR2 comprising SEQ ID NO: 5, wherein Xl is T or S; x2 is L, V or I; and

a light chain CDR3 comprising SEQ ID NO: 6, or consists of, an amino acid sequence M-X1-H-X2-E-Y-P-X3-T-F, wherein X1 is Q or N; x2 is I, V or L; x3 is L or I, preferably L,

for example, the antibody may comprise a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDRI, a light chain CDR2, and a light chain CDR3 comprising the combinations of amino acid residue substitutions shown in the following table:

i) the antibodies comprise combinations of amino acid residue substitutions shown in the following table:

ii) the antibody in i) further comprises the amino acid residue substitution combinations shown in the following table:

wherein the antibody is represented by the formula 10^-9M or less KD binds to HRP-II protein.

In some embodiments, the invention provides an antibody, or antigen-binding fragment thereof, that binds HRP-II, wherein the antibody comprises:

a heavy chain CDR1 comprising SEQ ID NO: 21 or consists of the amino acid sequence shown in 21;

a heavy chain CDR2 comprising SEQ ID NO: 22 or consists thereof; and

a heavy chain CDR3 comprising SEQ ID NO: 23 or consists thereof;

and the antibody further comprises:

a light chain CDRl comprising SEQ ID NO: 24 or consists thereof;

a light chain CDR2 comprising SEQ ID NO: 25 or consists thereof; and

a light chain CDR3 comprising SEQ ID NO: 26 or consists thereof.

In some embodiments, the antibodies of the invention may further comprise the framework regions FR-H1, FR-H2, FR-H3 and FR-H4 of the heavy chain variable region, and the framework regions FR-L1, FR-L2, FR-L3 and FR-L4 of the light chain variable region, wherein

FR-H1 comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence identical to SEQ ID NO: 27, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 27 compared to or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions);

FR-H2 comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence identical to SEQ ID NO: 28, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 compared to or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions);

FR-H3 comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence substantially identical to SEQ ID NO: 29, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 29 compared to or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions);

FR-H4 comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence identical to SEQ ID NO: 30, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30 compared to or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions); and

FR-L1 comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence substantially identical to SEQ ID NO: 31, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31 compared to or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions);

FR-L2 comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence substantially identical to SEQ ID NO: 32, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 32, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-L3 comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence identical to SEQ ID NO: 33, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 33, or consists of, one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in seq id no;

FR-L4 comprises the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence corresponding to SEQ ID NO: 34, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 34 compared to or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions).

(i) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 17, or

And SEQ ID NO: 17 has at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or

And SEQ ID NO: 17, and an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in seq id no, and

(II) a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 19, or

And SEQ ID NO: 19, or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence depicted in seq id No. 19, or

And SEQ ID NO: 19, or a variant thereof, wherein said variant has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to the amino acid sequence depicted in seq id no.

In some embodiments, the antibodies of the invention are present at 10^-10M or less KD binds to HRP-II protein.

In some embodiments, an antibody of the invention binds HRP-II protein with an EC50 of less than about 100nM, e.g., less than about 10nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, or less.

In some embodiments, antigen-binding fragments of the antibodies of the invention may include Fab, Fab ', F (ab')₂Fd, Fv, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.

In some embodiments, the present invention provides an isolated polypeptide selected from the group consisting of:

(1) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 21, 22 and 23, wherein the polypeptide specifically binds HRP-II as part of an anti-HRP-II antibody further comprising a sequence set forth in SEQ ID NO: 24, 25 and 26;

(2) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 24, 25 and 26, wherein the polypeptide specifically binds HRP-II as part of an anti-HRP-II antibody further comprising a sequence set forth in SEQ ID NO: 21, 22 and 23;

(3) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an anti-HRP-II antibody that specifically binds HRP-II, said antibody further comprising SEQ ID NO: 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence;

(4) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an anti-HRP-II antibody that specifically binds HRP-II, said antibody further comprising SEQ ID NO: 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence;

(5) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an anti-HRP-II antibody that specifically binds HRP-II, said antibody further comprising SEQ ID NO: 20 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence;

(6) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is part of an anti-HRP-II antibody that specifically binds HRP-II, said antibody further comprising SEQ ID NO: 18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations (preferably conservative mutations, preferably substitutions, insertions or deletions) compared to said sequence.

In some embodiments, the invention provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof or an isolated polypeptide of the invention.

In some embodiments, the invention provides a vector comprising an isolated polynucleotide described herein.

In some embodiments, the invention provides a host cell comprising an isolated polynucleotide or vector described herein.

In some embodiments, the invention provides a method of making an antibody or antigen-binding fragment thereof described herein, the method comprising culturing a host cell described herein.

In some embodiments, the invention provides antibody conjugates comprising an antibody or antigen-binding fragment thereof described herein and a coupling moiety coupled thereto, preferably, the coupling moiety comprises a label selected from the group consisting of a purification tag (e.g., a His-tag), a detectable label, such as colloidal gold, a radiolabel, a luminescent material, a colored material, an enzyme, such as a fluorescent label, a chromophoric label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and derivatives thereof, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, a spin label.

In some embodiments, the invention provides a kit or diagnostic agent comprising an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein described herein, wherein:

1) preferably, the kit further comprises an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein that binds a Plasmodium antigen other than HRP-II, such as a Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and/or Plasmodium ovale-specific antigen or a consensus antigen, such as an aldolase, such as lactate dehydrogenase PLDH, such as Plasmodium lactate dehydrogenase PLDH or Plasmodium species-specific lactate dehydrogenase PLDH,

2) preferably, the kit further comprises one or more additional antibodies that specifically recognize an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein described herein, and/or an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein that specifically recognizes an antigen of a plasmodium other than HRP-II, optionally the one or more additional antibodies further comprise a detectable label, such as colloidal gold, a radiolabel, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and derivatives thereof, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, a spin label;

3) optionally, the kit is a Rapid Diagnostic (RDT) kit, e.g., a kit employing thin film immunochromatography of colloidal gold methods, e.g., comprising a solid support, e.g., a membrane support, to which antibodies or antigen-binding fragments thereof, antibody conjugates, or fusion proteins that bind a plasmodium antigen and optionally a control antibody are immobilized;

4) optionally, the kit is an enzyme-linked immunosorbent (ELISA) kit.

In some embodiments, the invention provides the use of an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein described herein in the preparation of a kit for 1) detecting the presence or level of HRP-II in a sample, 2) diagnosing plasmodium falciparum infection, and/or 3) differentially diagnosing plasmodium falciparum infection from other plasmodium falciparum infections.

In some embodiments, the source of the sample is not particularly limited, and may include, for example, a tissue, cell, or fluid sample, such as a sample of a bodily fluid, e.g., cerebrospinal fluid, urine, saliva, blood sample. In order to avoid the defects of the traditional hybridoma technology, the invention provides an expression vector of an anti-plasmodium falciparum HRP-II monoclonal antibody, and provides an anti-plasmodium falciparum HRP-II monoclonal antibody sequence, which is used for expressing the anti-plasmodium falciparum HRP-II monoclonal antibody through a recombination technology and is used for diagnosing plasmodium falciparum.

In some embodiments, the invention includes one or more of the following aspects:

1) cloning of an anti-HRP-II monoclonal antibody light chain gene;

2) cloning the heavy chain gene of the anti-HRP-II monoclonal antibody;

3) constructing an expression vector of the anti-HRP-II monoclonal antibody;

4) aiming at the expression vector in the step 3), obtaining an Anti-HRP-II monoclonal antibody by adopting an expression system such as a eukaryotic expression system such as a CHO eukaryotic expression system;

5) aiming at the step 4), performing activity identification and affinity analysis on the Anti-HRP-II monoclonal antibody.

Drawings

FIG. 1: purified antibody reducing SDS-PAGE patterns, wherein lanes 1, 2, and 3 show 5. mu.g antibody reducing SDS-PAGE patterns obtained by purification on a ProteinA affinity column.

Detailed Description

The embodiments of the present invention are explained in detail below, and the described embodiments are not restrictive and may be combined with each other.

The term "amino acid" refers to naturally occurring or non-naturally occurring carboxy α -amino acids the term "amino acid" as used herein may include naturally occurring and non-naturally occurring amino acids including alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). non-naturally occurring amino acids include, but are not limited to, α -aminoadipic acid, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoproline, homosarcosine, homohistidine, etc.

Peptides, polypeptides, proteins are not strictly distinguished herein and may be used interchangeably in some instances, generally referring to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic.

Amino acid residues with similar side chains are known in the art and include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

In the case of two amino acid sequences, the sequences of the invention encompass corresponding sequences having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70% sequence identity to the sequences shown in the sequence listing when optimally aligned, such as by the programs GAP or BESTFIT using default GAP weights. For example, in some embodiments, an antibody of the invention comprises an amino acid sequence identical to SEQ ID NO: 1-3, and SEQ ID NO: 4-6, or the light chain CDR of SEQ ID NO: 21-23, and SEQ ID NO: 24-26, and a light chain CDR having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70% sequence identity to the corresponding heavy and light chain CDRs. For example, in some embodiments, an antibody of the invention comprises an amino acid sequence identical to SEQ ID NO: 27-30, heavy chain FR1-4, SEQ ID NO: 31-34, light chain FR1-4 having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70% sequence identity to the corresponding heavy chain FR1-4 and light chain FR 1-4. For example, in some embodiments, an antibody of the invention comprises an amino acid sequence identical to SEQ ID NO: 17, the heavy chain variable region of SEQ id no: 19, and an antibody to the heavy chain variable region and the light chain variable region having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70% sequence identity to the corresponding light chain variable regions. For example, in some embodiments, an antibody of the invention comprises an amino acid sequence identical to SEQ id no: 18, SEQ ID NO: 20, and a light chain variable region comprising at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70% sequence identity to the corresponding heavy and light chains. In some embodiments, the invention encompasses antigen-binding fragments, antibody conjugates, or fusion proteins of the antibodies. In some embodiments, the invention also includes corresponding isolated polypeptides comprising the heavy and/or light chains of the above-described antibodies. In some embodiments, the invention includes polynucleotides, vectors, host cells encoding the antibodies and isolated polypeptides.

In some embodiments, the residues of the antibodies of the invention that are not identical differ by conservative amino acid substitutions. Sequence identity can be measured using sequence analysis software, as known to those skilled in the art. For example, publicly available GCG software contains programs such as "Gap" and "BestFit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides or between a wild-type protein and its mutant protein. Polypeptide sequences can also be compared using FASTA or ClustalW, using default or recommended parameters. The program in GCG version 6.1, FASTA (e.g., FASTA2 and FASTA3) gives the alignment and percent sequence identity of the best overlapping region between query and search sequences. Another algorithm is the computer program BLAST, especially blastp, using default parameters.

The term "antibody" is used herein in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments so long as they exhibit the desired biological activity, e.g., specifically bind to an HRP-II antigen or fragment thereof. "antibody fragments" include portions of full-length antibodies, preferably the antigen-binding or variable regions thereof. Examples of antibody fragments include Fab,Fab′，F(ab′)₂fd, Fv, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies. In some embodiments, the invention includes fusion proteins of antibodies or antibody fragments, e.g., fusion of a detectable label or tag, such as GFP, HA, Flag, His, GST, Myc, etc., may also be fused to other polypeptides suitable for facilitating antigen detection.

In some embodiments, the invention includes a nucleic acid sequence comprising an antibody or fragment thereof encoding anti-plasmodium falciparum HRP-II. Herein, a nucleic acid sequence comprises conservatively substituted variants thereof (e.g., substitution of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and encompass genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

In some embodiments, the invention includes an expression vector comprising a nucleic acid sequence encoding an antibody against plasmodium falciparum HRP-II or a fragment thereof, wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the coding sequence is linked to the regulatory sequences in a manner that allows for expression of the coding sequence. Regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell and include promoters, enhancers and other expression control elements.

Herein, a vector includes a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, which may be propagated and in which the genetic information may be present or absent) or an expression vector (i.e., a vector which comprises the necessary genetic elements to permit expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. In some embodiments, the vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. In some embodiments, the vector comprises a sequence carrying a gene of interest and a selectable marker (e.g., Dhfr, GS marker or resistance gene neo, hygro, etc.). In some embodiments, the vectors of the invention include 1) monocistronic vectors, such as antibodies for heavy and light chains using different vectors, co-transfecting host cells; or the heavy chain and light chain genes can be connected in series to the same vector, and the transcription and translation are respectively carried out by using respective promoters and terminators; 2) a bicistronic vector, for example, a ribosome internal entry site is constructed in the vector, and light chain and heavy chain genes are connected in series at two ends of the vector, so that the simultaneous transcription and translation of the light chain and the heavy chain are realized; 3) trans-complementation vectors, such as two vectors expressing light chain and heavy chain, respectively, contain different sequences encoding selectable marker genes, and the selectable marker can be correctly expressed only when the light chain and the heavy chain are transfected simultaneously, so that the selection efficiency is improved. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

The expression vectors of the invention are useful for transforming host cells. Such transformed cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the polypeptides of the invention. Host cells of the invention include any engineered cell suitable for the production of recombinant antibodies. In some embodiments, the host cell may comprise a microorganism such as a bacterium (e.g., e.coli, bacillus, etc.), a multicellular organism such as a yeast cell, an insect cell, a plant cell, or a mammalian cell, preferably a cell from a human. In some embodiments, the host cell may be used to produce an antibody described herein, such as a single chain antibody or antibody fragment, or an isolated polypeptide, fusion protein, or the like. In some embodiments, the host cells of the invention include mammalian cells suitable for the production of recombinant antibodies, such as CHO, myeloma cells, hybridoma cells, PerC.6, HEK293, NS0, sp2/0, and the like. In some embodiments, the host cell preferably comprises a cell that is good in growth, high in serum-free suspension culture density, strong in heterologous expression, and has the ability to be correctly post-translationally modified.

In some embodiments, the invention provides methods for the preparation of antibodies, antigen-binding fragments, variants and functional derivatives against plasmodium falciparum HRP-II. In some embodiments, the anti-plasmodium falciparum HRP-II monoclonal antibody is expressed by recombinant technology. In some embodiments, a host cell is transfected with a nucleic acid vector encoding an anti-plasmodium falciparum HRP-II antibody and cultured under suitable conditions to express the anti-plasmodium falciparum HRP-II antibody. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of an antibody against Plasmodium falciparum HRP-II. In some embodiments, when the antibodies of the invention are recombinantly produced, the expression product may be exported into the culture medium or carried on the surface of the transformed cells. Antibodies against Plasmodium falciparum HRP-II may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis. In some embodiments, the immunogen used to prepare the antibody may comprise whole Plasmodium falciparum HRP-II, or a fragment or derivative thereof. Preferred immunogens comprise all or part of Plasmodium falciparum HRP-II.

In some embodiments, antibodies described herein, such as antibodies against plasmodium falciparum HRP-II, can be used to detect the presence of one or more target molecules, such as HRP-II, in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. In some embodiments, the biological sample comprises a cell or tissue.

In some embodiments, methods for diagnosis or detection are provided, the methods comprising contacting a biological sample with an antibody described herein, such as an antibody against plasmodium falciparum HRP-II, under conditions that allow binding of the antibody to the target, and detecting whether a complex is formed between the antibody and the target. In some embodiments, the method may be an in vitro or in vivo method. The invention also relates to a method for diagnosing malaria in a subject potentially infected with plasmodium falciparum, comprising contacting a biological sample from said subject with an antibody of the invention under conditions such that the antibody forms an antigen/antibody complex with a target potentially present in said biological sample, and detecting the antigen/antibody complex potentially formed. In this method, in vitro diagnosis can be performed by ELISA assay. In some embodiments, the methods of the invention may further comprise contacting the biological sample with one or more antibodies diagnostic for other plasmodium antigens, which may be, for example, LSA-1, LSA-3, LSA-5, SALSA, STARP, TRAP, PfEXP1, CS, MSP-3-1, MSP-3-2, MSP-3-5, MSP-3-6, MSP1, MSP2, MSP4, MSP5, AMA-1, SERP, and GLURP.

In some embodiments, exemplary labels include, but are not limited to, radioisotopes, fluorophores, rhodamine labels, luciferase, luciferin, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labels, phage labels, and the like.

In some embodiments, the anti-plasmodium falciparum HRP-II antibodies or fragments thereof of the present invention can be used as antigens in immunoassays and corresponding kits for detection of HRP-II antibodies. In some embodiments, immunoassays of the invention include ELISA, indirect immunofluorescence assays IFA, and radioimmunoassay RIA, as well as other non-enzyme-linked antibody binding assays or methods.

In some embodiments, for example in an ELISA double antigen sandwich protocol, the HRP-II antibody may be coated on the microplate, the HRP-II antigen in the sample captured, and then re-bound to the antigen bound on the plate with the labeled antibody, and the results read after development. In some embodiments, the HRP-II antibodies of the present invention may be used to coat a microplate or as a labeled secondary antibody. In some embodiments, the antibody or fragment thereof against plasmodium falciparum HRP-II is immobilized to a surface, e.g., a solid support, e.g., a plastic, a membrane such as nitrocellulose, a glass, or a metal support. In some embodiments, the solid support can be in the form of a strip or a card. In some embodiments, a sample from a subject is contacted with the solid support and then contacted with an antibody indicator labeled with a detectable label, such as a rhodamine label, for development. In some embodiments, non-specific sites can be blocked with blocking agents such as bovine serum albumin, milk powder solutions, gelatin, PVP, Superblock, thus reducing the background caused by non-specific binding. In some embodiments, the antiserum may be diluted with a diluent, such as BSA and Phosphate Buffered Saline (PBS)/Tween, to help reduce non-specific background.

In some embodiments, this document relates to methods for determining the presence or quantity of an HRP-II antigen. In some embodiments, the method comprises contacting a biological sample that may contain an HRP-II antigen with an anti-plasmodium falciparum HRP-II antibody of the invention, and quantitatively or qualitatively determining binding between the at least one anti-plasmodium falciparum HRP-II antibody and the antigen in the sample. The method may take any form and may be, for example, a non-competitive or competitive ELISA, RIA or magnetic immunoassay, agglutination assay and surface plasmon resonance based assay such as Biacore assay.

In this context, a biological sample may refer to a sample of biological tissue, cells or fluid, such as a body fluid, in a healthy and/or pathological state. In some embodiments, the biological sample is from a subject suspected of being infected with malaria. In some embodiments, the biological sample may be a bodily fluid sample, such as a cerebrospinal fluid, urine, saliva, blood, or the like sample.

In some embodiments, provided herein are test or diagnostic devices or related kits that may include:

(i) an anti-plasmodium falciparum HRP-II antibody capable of binding to an HRP-II antigen; and

(II) an indicator that indicates when the antigen binds to the anti-plasmodium falciparum HRP-II antibody.

In some embodiments, the kit may also include antibodies to other antigens of plasmodium in addition to antibodies to plasmodium falciparum HRP-II of the invention, e.g., for identifying different plasmodium infections. In some embodiments, the other antigen includes, for example, plasmodium falciparum, plasmodium vivax, plasmodium malariae, and/or plasmodium ovale specific or consensus antigens, such as aldolases, e.g., lactate dehydrogenase PLDH, e.g., plasmodium lactate dehydrogenase PLDH or plasmodium species specific lactate dehydrogenase PLDH, e.g., plasmodium glutamate dehydrogenase PGDH, LSA-1, LSA-3, LSA-5, SALSA, STARP, TRAP, PfEXP1, CS, MSP-3-1, MSP-3-2, MSP-3-5, MSP-3-6, MSP1, MSP2, MSP4, MSP5, AMA-1, SERP, and GLURP.

In some embodiments, the kit may include a test strip or card onto which the liquid sample from the subject is placed, or an ELISA assay plate having wells in which liquid samples from individual subjects can be placed. In some embodiments, the kit may include a testing device configured for use in a flow cytometer, a bioanalyzer, a biosensor.

In some embodiments, the kit is a Rapid Diagnostic (RDT) kit, e.g., a kit that employs thin film immunochromatography, e.g., a kit that employs colloidal gold methods, e.g., comprising a solid support, e.g., a membrane support, to which antibodies or antigen-binding fragments thereof, antibody conjugates, or fusion proteins that bind a plasmodium antigen and optionally a control antibody are immobilized. In some embodiments, the kit, such as a Rapid Diagnostic (RDT) kit, may include a test strip or card that may include a sample pad, a labeled pad adjacent to the sample pad containing plasmodium antibodies (which may include one or more antibodies, such as antibodies to plasmodium falciparum HRP-II described herein and antibodies to other antigens of plasmodium, which may have a detectable label such as colloidal gold), a membrane, such as a cellulose membrane, adjacent to the labeled pad, and an adjacent bibulous pad. In some embodiments, the membrane may be provided with a detection line and a quality control line separated from each other, the detection line containing a plasmodium-specific antibody binding to a different epitope from the labeled antibody, and the quality control line may contain an antibody capable of specifically binding to the labeled antibody. In some embodiments, the test strip or card can include a support, a sample pad, a label pad, a membrane such as a nitrocellulose membrane, an absorbent pad, one or more detection lines, and a quality control line, the sample pad, the labeling pad, the nitrocellulose membrane, and the absorbent pad are sequentially disposed from one end to the other end of the support, the sample pad partially overlaps the label pad portion, the label pad partially overlaps the nitrocellulose membrane, the nitrocellulose membrane is overlapped with the absorption pad part, the one or more detection lines and the quality control line are respectively arranged on the nitrocellulose membrane at intervals, the labeling pad may be coated with a detectable label such as a colloidal gold-labeled plasmodium antibody, the one or more detection lines may contain plasmodium-specific antibodies that bind to different epitopes from the labeled antibody, and the quality control line may contain an antibody that specifically binds to the labeled antibody.

In some embodiments, the invention provides a kit comprising materials useful for diagnosing plasmodium infection. In some embodiments, the kit comprises a container and a label or package insert on or with the container. In some embodiments, suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials such as glass or plastic. The container contains a composition, either alone or in combination with another composition effective for diagnosing plasmodium infection. At least one active agent in the composition is an antibody against Plasmodium falciparum HRP-II herein. Further, the kit may comprise: (a) a first container comprising a composition therein, wherein the composition comprises an antibody to plasmodium falciparum HRP-II herein; and (b) a second container having a composition contained therein, wherein the composition comprises a second antibody and/or other related reagents. In some embodiments, antibodies to other antigens of malaria in addition to antibodies to plasmodium falciparum HRP-II of the invention may be included in the kit, for example to identify different plasmodium infections. The kit of the invention may further comprise a package insert indicating that the composition may be used for diagnosing the disease or infection. The kit may also include a second or third container containing a buffer, such as water for injection, phosphate buffered saline, glucose solution, and may also include other materials, such as other buffers, diluents, filters, needles, and syringes.

In some embodiments, provided herein are kits, such as microtiter plates (onto which the anti-plasmodium falciparum HRP-II antibody may be coated), comprising the anti-plasmodium falciparum HRP-II antibody. In some embodiments, the microtiter plate may comprise titer wells, such as polystyrene microtiter wells, which are coated with an antibody against plasmodium falciparum HRP-II, or with plasmodium infected whole cell runs, or with cell lysates thereof.

In some embodiments, provided herein are kits for determining, for example, a subject infected with plasmodium, the kit comprising at least one anti-plasmodium falciparum HRP-II antibody of the invention, an associated buffer, reagents required for reacting a liquid sample with the anti-plasmodium falciparum HRP-II antibody, and reagents for determining the presence of a positive or negative binding reaction between the antibody and the anti-plasmodium falciparum HRP-II antibody. Such a kit may include a container for separating and/or storing a liquid sample, containers and reagents for contacting the anti-plasmodium falciparum HRP-II antibody with the sample, and reagents for determining the presence of binding between the anti-plasmodium falciparum HRP-II antibody and components in the sample. For determining the presence of the antibody, the kit may for example utilize an antibody or antigen bearing a second label (double antigen sandwich), wherein the label may be any suitable label, such as a fluorescent or radioactive label, an enzymatic label or a conjugated label, such as a biotin label for binding to streptavidin. In the kit, the antibody against plasmodium falciparum HRP-II may be bound to a solid support, or the kit may include at least a solid support suitable for binding the antibody against plasmodium falciparum HRP-II.

In some embodiments, the HRP-II antibody of the kit may be in the form of a liquid solution, attached to a solid support, or as a dry powder. When the HRP-II antibody reagent is a liquid solution, the liquid solution may be an aqueous solution. When the reagent is in a form attached to a solid support, the solid support may preferably be a chromatographic medium such as a membrane, test strip, plastic bead or plate, or a microscope slide. When the reagent is a dry powder, the powder may be reconstituted by addition of a suitable solvent. In some embodiments, the kit may further comprise a container comprising a suitable solvent.

In some embodiments, the kit includes a container comprising a quantity of a second antibody, such as an alkaline phosphatase-conjugated second antibody, and a second container comprising a quantity of a buffer. In other embodiments, the kit may further comprise a third container comprising a suitable substrate, such as PNPP for alkaline phosphatase, or a substrate for peroxidase. The fourth container may include a suitable "stop" buffer.

Examples

1. Expression plasmid construction

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART^TMRACE cDNA amplification kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation. Hybridoma cell strain secreting Anti-HRP-II 6F5 monoclonal antibodyThe hybridoma cell strain is obtained by fusing and screening spleen cells of a MA-HRPII Ag protein (MyBioSource, MBS319418) immunized mouse and SP2/0 cells, rapidly recovering the cells from liquid nitrogen, and adding the cells into a complete culture medium (20% fetal bovine serum + 80% RPMI 1640) for culture for later use.

1.1 primers

Amplification of Heavy Chain and Light Chain 5' RACE primers:

SMARTER II A Oligonucleotide：5’＞AAGCAGTGGTATCAACGCAGAGTACXXXXX＜3’(SEQ ID NO.7)；

5′-RACE CDS Primer(5′-CDS)：5’＞(T)₂₅VN＜3’(N＝A，C，G，orT；V＝A，G，orC)(SEQ ID NO.8)；

Universal Primer A Mix(UPM)：5’＞CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT＜3’(SEQ ID NO.9)

Nested Universal Primer A(NUP)：5’＞AAGCAGTGGTATCAACGCAGAGT＜3’(SEQ IDNO.10)

mIg-kR：5’＞CTAACACTCATTCCTGTTGAAGCTCTTGACAAT＜3’(SEQ ID NO.11)。

mIg-HR：5’＞TCATTTACCAGGAGAGTGGGAGAGGC＜3’(SEQ ID NO.12)。

1.2 cloning and sequencing of antibody variable region genes

RNA was extracted from hybridoma cell lines secreting Anti-HRP-II 6F5 monoclonal antibody using SMARTER according to the manufacturer's recommended protocol^TMThe RACE cDNA Amplification Kit and SMARTER II AOligonucleotide and 5' -CDS Primer in the Kit are used for first strand cDNA synthesis, the obtained first strand cDNA product is used as a PCR Amplification template, the Light Chain gene is amplified by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mkR Primer, the Heavy Chain gene is amplified by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mHR Primer, the Primer pair of the Light Chain amplifies a target band about 0.7KB, the Primer pair of the Heavy Chain amplifies a target band about 1.4KB, the products are purified and recovered by agarose gel electrophoresis, the products are inserted into pMD-18T vector after being subjected to A reaction by rTaq DNA polymerase and are transformed into DH5 α competent cells,after colonies were grown, 9 clones of the Heavy Chain and Light Chain gene clones were transferred to Invitrogen corporation for sequencing.

1.3 sequence analysis of Anti-HRP-II 6F5 antibody variable region Gene

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 399bp, belongs to VkII gene family, and a leader peptide sequence of 60bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the HeavyChain primer pair, the VH gene sequence is 411bp, belongs to a VH1 gene family, and has a leader peptide sequence with the front part of 57 bp. The antibody sequence is shown in SEQ ID NOs: 35-38.

1.4 construction of recombinant antibody expression plasmid

pcDNA^TM3.4

vector is a constructed eukaryotic expression vector of a recombinant antibody and is purchased from Thermoscientific. The expression vector has introduced multiple cloning enzyme cutting sites of HindIII, BamHI, EcoRI and the like, and is named as pcDNA3.4A expression vector, and the subsequent expression vector is called as 3.4A expression vector for short; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of Anti-HRP-II 6F5 are designed, two ends of the primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and the primers are as follows:

HRP-II 6F5-HF：5’＞CCCAAGCTTGCCACCATGGAATGGCCTTGTATCTTTCTCTTCCTC＜3’(SEQ ID NO.13)；

HRP-II 6F5-HR：5’＞CCCGAATTCTCATTACTTGCCAGGAGAGTGGCTCAGGGACTTCTC＜3’(SEQ ID NO.14)；

HRP-II 6F5-LF：5’＞CCCAAGCTTGCCACCATGGAGTCACAGATTCAGGTCTTTGTATTCG＜3’(SEQ ID NO.15)；

HRP-II 6F5-LR：5’＞CCCGAATTCTCATTAACATTCATTTCTGTTAAAAGATTTGAC＜3’(SEQID NO.16)；

720bp Light Chain gene fragment and 1386bp Heavy Chain gene fragment are amplified from the cloned gene by a PCR amplification method. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2. Recombinant antibody production

2.1 transient transfection of recombinant antibody expression plasmid into CHO cells and identification of antibody activity of expression supernatant

2.1.1 transient transfection of recombinant plasmids into CHO cells

The 3.4A vector plasmid containing the antibody light and heavy chain DNA constructed in example 1 was diluted to 400. mu.g/ml with ultrapure water, and CHO cells 1.7 × 10 were adjusted⁷cells/ml in a centrifuge tube, 100. mu.l plasmid mixed with 700. mu.l cells, transferred to an electric rotor, transferred to 10ml of medium containing CD CHO AGT (Thermo Scientific), cultured in a shaker at 37 ℃ (8% CO)₂The amplitude is 115-200 rpm); sampling every day to detect the cell viability, and centrifuging the cell culture supernatant when the cell viability is lower than 50%.

2.1.2 characterization of antibody Activity of expression supernatants

Diluting HRP-II protein with 50mM carbonate buffer solution to the specified concentration, 100uL per well, and coating overnight at 4 ℃; on the next day, washing with PBST washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), incubating at 37 deg.C for 1h with 120uL of blocking solution per well, and patting to dry; adding diluted cell supernatant at a multiple ratio, incubating at 37 deg.C for 30min (partial supernatant for 1h) at 100 uL/well; washing with PBST washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (Abcam, ab97019) produced by the company, 100uL of each well, 37 ℃ and 30 min; washing with PBST washing solution for 5 times, and drying; adding carbamide peroxide (50 uL/hole) and tetramethylbenzidine (50 uL/hole) for developing for 10 min; adding dilute hydrochloric acid to terminate the reaction, wherein the concentration is 50 uL/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader.

Note: the reference substance is sequentially diluted by 2 times from 100ng/ml, and no reference substance is present in blank wells

From the above table results, the antibody generated after the constructed 3.4A recombinant expression plasmid was transiently transformed was active against HRP-II protein.

2.2 antibody purification and affinity analysis, Activity identification

2.2.1 ProteinA affinity chromatography column purification of expression supernatant

The supernatant of the fermentation broth was collected, filtered through a 0.22 μ M membrane, and subjected to column hanging under a constant flow rate through a column packed with MabSlelect SureLX (GE Healthcare) affinity, and then eluted with 20mM NaAc (pH3.4) solution, and a predetermined amount of 1M Tris solution was added to the sample collection tube for preneutralization. The eluted sample was dialyzed against PBS (pH7.4) for three changes to obtain purified antibody. Mu.g of the purified antibody was subjected to reducing SDS-PAGE. The results are shown in FIG. 1, and lanes 1, 2 and 3 are all 5ug loading.

2.2.2 affinity analysis of purified antibodies

Performing enzyme-free indirect method data in the same way of activity identification, and performing four gradient of 0.5ug/ml, 0.25ug/ml, 0.125ug/ml and 0.0625 ug/ml; the antibody was diluted in a 2-fold gradient starting at 100ng/ml to 0.195ng/ml loading. And obtaining the OD values corresponding to different antibody concentrations under different coating concentrations. Under the same coating concentration, the antibody concentration is used as an abscissa and the OD value is used as an ordinate, logarithmic mapping is carried out, and the antibody concentration at 50% of the maximum OD value is calculated according to a fitting equation; substitution into the formula: k ═ n-1)/(2 × (n × Ab '-Ab)) the reciprocal of the affinity constant was calculated, where Ab and Ab' respectively represent the antibody concentration at 50% of maximum OD value at the corresponding coating concentration (Ag, Ag '), and n ═ Ag/Ag'; every two coating concentrations can be combined to calculate a K value, six K values can be obtained finally, the average value of the K values is taken, and the reciprocal value is calculated to be the affinity constant KD.

Affinity analysis data of purified Anti-HRP-II 6F5 monoclonal antibody

Sample name	KD
		Anti-HRP-II 6F5	5.216E-10

2.2.3 Activity characterization of purified antibodies

Diluting MA-HRPII Ag recombinant MA protein (MyBioSource, MBS319418) (ex 150520-1) to 1ug/ml with 50mM carbonate buffer solution coating solution for microplate coating, wherein each well is 100uL, and the temperature is 4 ℃ overnight; the next day, the wash solution was washed 2 times with PBST and patted dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1h and 120uL per well; adding diluted Anti-HRP-II 6F5MA monoclonal antibody, diluting by 5 times from 1000ng/ml, loading, 100 uL/well, 37 deg.C, 30min (partial supernatant for 1 h); washing with PBST washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100uL per well at 37 ℃ for 30 min; washing with PBST washing solution for 5 times, and drying; adding carbamide peroxide (50 uL/hole) and tetramethylbenzidine (50 uL/hole) for developing for 10 min; adding dilute hydrochloric acid to terminate the reaction, wherein the concentration is 50 uL/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader.

Activity identification of purified Anti-HRP-II 6F5 monoclonal antibody

Sample concentration ng/ml	1000	200	40	8	1.6	0.32	0
								Anti-HRP-II 6F5	2.246	2.327	2.034	0.693	0.197	0.062	0.049

Provides an Anti-plasmodium falciparum HRP-II monoclonal antibody sequence, obtains an Anti-HRP-II monoclonal antibody with activity and higher affinity by a recombinant antibody technology, and has the following advantages:

1) compared with the hybridoma technology, the CHO cell strain is more stable, is not easy to lose the secretion capacity of the antibody in the process of passage, freezing storage and recovery, and is easier to store the precious cell strain.

2) The expression vector of the anti-HRP-II recombinant antibody is successfully constructed, can be expressed in CHO cells and has biological activity. Compared with the hybridoma technology, the recombinant antibody expression yield of the CHO cell strain can reach about 2g/L, and the cost is lower during mass production; in addition, the cell strain is more stable, the production stability is good, the batch difference is smaller, the purification difficulty is smaller, and the cost is lower.

3) By adopting a recombinant antibody technology, an expression vector capable of specifically recognizing the plasmodium falciparum HRP-II protein epitope is constructed, a specific monoclonal antibody is produced, the occurrence of non-specific reaction is reduced, and the specificity of detecting the plasmodium falciparum is improved.

3. The light chain CDRs and heavy chain CDRs of the antibodies obtained in the above examples (having light and heavy chains with sequences shown in SEQ ID NOS: 20 and 18 as WT) were mutated.

Upon analysis, the complementarity determining region (WT) of the heavy chain:

CDR-VH1 is G-Y-X1(S) -F-X2(S) -S-Y-W-X3(L) -H;

CDR-VH2 is N-I-Y-X1(A) -G-S-X2(R) -F-T-X3(Q) -Y-N-E;

CDR-VH3 is G-X1(L) -X2(F) -Y-G-S-X3(N) -G-D-F;

complementarity determining regions of the light chain:

CDR-VL1 is S-K-S-X1(L) -X2(L) -Q-T-N-X3(P) -N-X4(S) -Y-L-Y;

CDR-VL2 is R-M-X1(S) -N-X2(I) -A-S;

CDR-VL3 is M-X1(N) -H-X2(V) -E-Y-P-X3(L) -T-F;

wherein, X1, X2, X3 and X4 are all mutation sites.

Mutation site design

Detecting the activity of the antibody after mutation, diluting MA-HRPII Ag protein (Fipeng organism, 150520-1) to 1ug/ml by coating solution, coating by a micropore plate at 100uL per hole and standing overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1h and 120uL per well; adding diluted Anti-HRP-II 6F5 monoclonal antibody, 100 uL/well, 37 ℃, 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100uL per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding carbamide peroxide (50 uL/hole) and tetramethylbenzidine (50 uL/hole) for developing for 10 min; adding dilute hydrochloric acid to terminate the reaction, wherein the concentration is 50 uL/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. Some of the results are as follows:

data of antibody Activity analysis

Antibody concentration (ng/ml)	WT	Mutation 1	Mutation 2	Mutation 3	Mutation 4	Mutation 5
							1000	2.246	2.413	2.123	0.803	0.498	-
200	2.327	2.495	2.010	0.325	0.075	-
							40	2.034	2.422	1.995	0.022	-	-
8	0.693	1.446	1.012	-	-	-
							1.6	0.197	0.386	0.264	-	-	-
0.32	0.062	0.175	0.103	-	-	-
							0	0.049	0.043	0.025	-	-	-

"-" indicates no activity.

As can be seen from the above table, the activity of mutations 1 and 2 is better, and particularly, the activity of mutation 1 is the best. Furthermore, mutation 1 is used as a framework sequence to screen mutation sites with better potency (ensuring that the activity of the screened antibody is similar to that of mutation 1 and the antibody activity is +/-10%), and partial results are as follows.

List of mutation sites with better potency

Affinity assay

Affinity assay data for screened mutant antibodies

Therefore, the affinity of the screened mutant site to the antibody has little influence. To verify the above results, the above experiment was repeated using WT as a backbone sequence, and affinity verification of the mutation site was performed, and some results are as follows.

Mutation with WT as backbone

Affinity assay data

	KD(M)
		WT	5.216E-10
WT 1-1	5.08E-10
		WT 1-2	1.58E-10
WT 1-3	5.99E-10
		WT 1-4	4.16E-10
WT 1-5	5.95E-10

From the above results, it was found that the mutation site was not greatly related to other sites while ensuring the antibody activity.

Description of related sequences:

CDR-VH1.G-Y-X1-F-X2-S-Y-W-X3-H(SEQ ID NO.1)

wherein:

x1 is S, Y or T;

x2 is S or T, preferably T;

x3 is L, V or I;

CDR-VH2.N-I-Y-X1-G-S-X2-F-T-X3-Y-N-E(SEQ ID NO.2)

wherein:

x1 is a or P, preferably P;

x2 is R or K;

x3 is N, R or Q;

CDR-VH3.G-X1-X2-Y-G-S-X3-G-D-F(SEQ ID NO.3)

wherein:

x1 is I or L, preferably I;

x2 is Y or F;

x3 is Q, R or N;

CDR-VL1.S-K-S-X1-X2-Q-T-N-X3-N-X4-Y-L-Y(SEQ ID N0.4)

wherein:

x1 is I or L;

x2 is L or I;

x3 is P or G, preferably G;

x4 is S or T;

CDR-VL2.R-M-X1-N-X2-A-S(SEQ ID NO.5)

wherein:

x1 is T or S;

x2 is L, V or I;

CDR-VL3.M-X1-H-X2-E-Y-P-X3-T-F(SEQ ID NO.6)

wherein:

x1 is Q or N;

x2 is I, V or L;

x3 is L or I, preferably L;

Claims

1. an antibody or antigen-binding fragment thereof that binds plasmodium falciparum histidine-rich protein-II, HRP-II, wherein the antibody comprises the following amino acid sequence or complementarity determining regions having at least 80% sequence identity thereto:

a heavy chain CDR1 comprising SEQ ID NO: 1, or consists of, the amino acid sequence G-Y-X1-F-X2-S-Y-W-X3-H, wherein X1 is S, Y or T; x2 is S or T, preferably T; x3 is L, V or I;

a heavy chain CDR2 comprising SEQ ID NO: 2, or consists of, wherein X1 is a or P, preferably P; x2 is R or K; x3 is N, R or Q; and

a heavy chain CDR3 comprising SEQ ID NO: 3, or consists of, wherein X1 is I or L, preferably I; x2 is Y or F; x3 is Q, R or N;

and the antibody further comprises:

a light chain CDRl comprising SEQ ID NO: 4, or consists of, an amino acid sequence S-K-S-X1-X2-Q-T-N-X3-N-X4-Y-L-Y, wherein X1 is I or L; x2 is L or I; x3 is P or G, preferably G; x4 is S or T;

a light chain CDR2 comprising SEQ ID NO: 5, or consists of, an amino acid sequence R-M-X1-N-X2-a-S, wherein X1 is T or S; x2 is L, V or I; and

a light chain CDR3 comprising SEQ ID NO: 6, or consists of, an amino acid sequence M-X1-H-X2-E-Y-P-X3-T-F, wherein X1 is Q or N; x2 is I, V or L; x3 is L or I, preferably L;

for example, the antibody may comprise a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDRl, a light chain CDR2, and a light chain CDR3 comprising the combinations of amino acid residue substitutions shown in the following table:

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

a heavy chain CDR2 comprising SEQ ID NO: 22 or consists thereof; and

a heavy chain CDR3 comprising SEQ ID NO: 23 or consists thereof;

and the antibody further comprises:

a light chain CDRl comprising SEQ ID NO: 24 or consists thereof;

a light chain CDR2 comprising SEQ ID NO: 25 or consists thereof; and

a light chain CDR3 comprising SEQ ID NO: 26 or consists thereof.

3. The antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the antibody further comprises the framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the heavy chain variable region, and the framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the light chain variable region, wherein

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody comprises:

(i) a heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO: 17, or

(II) a light chain variable region comprising or consisting of the sequence:

SEQ ID NO: 19, or

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody is present at 10^-10M or less KD binds to HRP-II protein.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody binds HRP-II protein with an EC50 of less than about 100nM, e.g., less than about 10nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM or less.

7. The method of any one of claims 1 to 6An antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, Fv, Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.

8. An isolated polypeptide selected from the group consisting of:

9. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the isolated polypeptide of claim 8.

10. A vector comprising the isolated polynucleotide of claim 9.

11. A host cell comprising the isolated polynucleotide of claim 9 or the vector of claim 10.

12. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising culturing the host cell of claim 11.

13. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7 and a coupling moiety coupled thereto, preferably the coupling moiety comprises a label selected from the group consisting of a purification label (e.g., a His-label), a detectable label, such as colloidal gold, a radiolabel, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophoric label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and derivatives thereof, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, a spin label.

14. A kit or diagnostic agent comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the antibody conjugate of claim 13, wherein:

1) preferably, the kit or diagnostic agent further comprises an antibody or antigen-binding fragment thereof, antibody conjugate, or fusion protein that binds a plasmodium antigen other than HRP-II including, for example, plasmodium falciparum, plasmodium vivax, plasmodium malariae, and/or plasmodium ovale specific antigen or consensus antigen, for example, aldolases, such as lactate dehydrogenase PLDH, such as plasmodium lactate dehydrogenase PLDH or plasmodium species specific lactate dehydrogenase PLDH, such as plasmodium glutamate dehydrogenase PGDH, plasmodium antigens LSA-1, LSA-3, LSA-5, SALSA, STARP, TRAP, PfEXPl, CS, MSP-3-1, MSP-3-2, MSP-3-5, MSP-3-6, MSP1, MSP2, MSP4, MSP5, AMA-1, AMA-3, or a fusion protein, The combination of the SERP and the GLURP,

2) preferably, the kit or diagnostic agent further comprises one or more additional antibodies that specifically recognize the antibody or antigen-binding fragment thereof of any one of claims 1-7, the antibody conjugate of claim 13, and/or the antibody or antigen-binding fragment thereof, the antibody conjugate, or the fusion protein that specifically recognizes the plasmodium antigen other than HRP-II, optionally, the one or more additional antibodies further comprise a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron dense label, such as a radioisotope, a fluorophore, rhodamine and derivatives thereof, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, a carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, a spin label,

4) optionally, the kit is an enzyme-linked immunosorbent assay (ELISA) kit, an indirect immunofluorescence assay (IFA) kit, or a Radioimmunoassay (RIA) kit.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the antibody conjugate of claim 13 in the preparation of a diagnostic agent or kit for 1) detecting the presence or level of HRP-II in a sample, 2) diagnosing plasmodium falciparum infection, and/or 3) differentially diagnosing plasmodium falciparum infection from other plasmodium falciparum infections.

16. The use of claim 15, wherein the sample comprises a tissue, cell or fluid sample, such as a sample of a bodily fluid, such as cerebrospinal fluid, urine, saliva, blood sample.