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WO2023035974A1 - Anticorps contre le plasmodium et son application - Google Patents

Anticorps contre le plasmodium et son application Download PDF

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Publication number
WO2023035974A1
WO2023035974A1 PCT/CN2022/115075 CN2022115075W WO2023035974A1 WO 2023035974 A1 WO2023035974 A1 WO 2023035974A1 CN 2022115075 W CN2022115075 W CN 2022115075W WO 2023035974 A1 WO2023035974 A1 WO 2023035974A1
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antibody
antigen
plasmodium
seq
binding fragment
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Chinese (zh)
Inventor
孟媛
钟冬梅
覃文新
黄玉玲
熊俊文
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the technical field of antibodies. More specifically, it relates to an anti-Plasmodium antibody and its application.
  • Malaria is a vector-borne infectious disease caused by the infection of Plasmodium through the bite of Anopheles mosquito or the blood transfusion of a person with Plasmodium. It is listed as a disease by the World Health Organization (WHO) together with AIDS and tuberculosis Three major global public health problems. According to the World Malaria Report 2019 released by WHO, there were 228 million malaria cases worldwide in 2018, of which 405,000 died from the disease. There are four main types of Plasmodium that parasitize the human body: Plasmodium falciparum (pf), Plasmodium vivax (pv), Plasmodium malariae (pm), Plasmodium ovale , po). Among the malaria parasites that infect humans, P. falciparum is the most pathogenic.
  • the sporozoites After the malaria parasite bites the human body mainly through the female Anopheles mosquito, the sporozoites first invade the liver cells and reproduce and develop in them. When the liver cells rupture, a large number of merozoites released quickly enter the blood circulation and invade the red blood cells; In the process of asexual reproduction, not only a large number of broken red blood cells cause anemia, but also release many metabolites as pyrogens, stimulating the body to produce a strong protective immune response. Clinical manifestations of malaria vary according to the course of the disease and can range from more common symptoms such as fever, chills, and headache to more severe symptoms including severe anemia, respiratory distress associated with metabolic acidosis, multiorgan function Failure and cerebral malaria, these severe changes are collectively referred to as severe malaria.
  • the current methods for detecting malaria parasites can be divided into four categories.
  • One is the direct detection of Plasmodium by microscope, which is currently the gold standard for clinical diagnosis of malaria. However, this method is time-consuming and laborious, and requires skilled technicians and certain experimental conditions.
  • the second is the detection of Plasmodium nucleic acid. Although this method has high sensitivity and specificity, it requires more complex equipment and technical conditions as support. It is not suitable as a routine detection method in malaria endemic areas, and it is difficult to promote and apply it at the grassroots level.
  • the third is to detect plasmodium pigments. This method requires professional detection instruments and is generally used for laboratory research, not for on-site detection.
  • the fourth is the immune reaction of antigen and antibody to detect malaria parasites.
  • This method is simple to operate, fast, and has good accuracy. At the same time, it has low requirements for laboratories and operators. It is generally applicable to grassroots departments such as hospital laboratory departments and disease control system laboratories. Screening plays an important role in the initial detection of malaria, in the successful control of outbreaks in hospitals and in the community, and in guiding treatment. Therefore, rapid immunodiagnostic products based on the detection of Plasmodium-specific antigens have received increasing attention in the diagnosis of malaria.
  • the commonly used malaria antigens are mainly Plasmodium falciparum-specific histidine-rich protein II (HRP-II) and Plasmodium lactate dehydrogenase (PLDH).
  • HRP-II is the most recognized diagnostic marker for Plasmodium falciparum, but the Plasmodium falciparum detection kit with HRP-II as the target antigen can only be used for the detection of Plasmodium falciparum, and cannot detect the widespread Plasmodium vivax; when the patient's blood After the live worms in the virus disappear, HRP-II will still exist in the human body for a long time, and the infection period cannot be accurately confirmed; the cross-reaction between anti-HRP-II monoclonal antibody and rheumatoid factor will cause false positive reaction.
  • Plasmodium lactate dehydrogenase is an important enzyme to ensure the normal progress of malaria parasite glycolysis. Compared with lactate dehydrogenase of human red blood cells and many other micro substances, it has significantly different physical and biochemical characteristics. It is a protein that must be expressed and has a high abundance, so it has become an important target for the detection of malaria parasites. Since PLDH is only produced by live malaria parasites, using PLDH as a method of detecting antigens can also identify the life and death of parasites in patients, so as to evaluate and monitor the therapeutic effect and relapse.
  • the PLDH produced by the four species of Plasmodium has different isomers, and has species- and genus-specific antigens, which can be mainly divided into two categories: one is species-specific PLDH, including pf PLDH, pv PLDH, pm PLDH, po PLDH, the monoclonal antibody produced with this as the target protein only recognizes the PLDH of a specific species of Plasmodium; The monoclonal antibody can recognize the PLDH of four kinds of Plasmodium.
  • Pan-PLDH monoclonal antibody products At present, there are few Pan-PLDH monoclonal antibody products on the market that can simultaneously detect P.
  • the technical problem to be solved in this disclosure is to overcome the defects of the specificity and sensitivity of the existing Pan-PLDH monoclonal antibodies for simultaneously detecting P. falciparum, P. vivax, P. ovale, and P.
  • the cloned antibody can simultaneously detect P. falciparum, P. vivax, P. ovale, and P. ovale, and has obvious advantages over existing mainstream materials in terms of antibody affinity, reactivity, specificity, and sensitivity.
  • Heavy chain CDR1 which comprises the amino acid sequence S-Y-T-M-H shown in SEQ ID NO.1, or consists of it;
  • a heavy chain CDR2 comprising or consisting of the amino acid sequence shown as H-I-N-P-S-S-G-Y-X1-N-X2-N-Q-K-F-X3-D, wherein X1 is I or L; X2 is I or L; X3 is K or Q; and
  • Heavy chain CDR3 which comprises the amino acid sequence T-G-T-G shown in SEQ ID NO.3, or consists of it;
  • antibody or antigen-binding fragment further comprises:
  • Light chain CDR1 which comprises the amino acid sequence T-A-S-S-S-V-S-S-S-G-Y-L-Q shown in SEQ ID NO.4, or consists of it;
  • Light chain CDR2 which comprises the amino acid sequence T-T-S-N-L-A-S shown in SEQ ID NO.5, or consists of it;
  • Light chain CDR3 which comprises the amino acid sequence H-Q-Y-H-R-S-P shown in SEQ ID NO.6, or consists of it.
  • Another object of the present disclosure is to provide nucleic acids, vectors or cells related to the antibody or antigen-binding fragment.
  • the present disclosure also provides methods of making the antibodies or antigen-binding fragments.
  • the present disclosure also provides an antibody conjugate, and a kit/diagnostic reagent comprising the above-mentioned antibody or antigen-binding fragment or the antibody conjugate.
  • the present disclosure also provides the use of the antibody or antigen-binding fragment or the antibody conjugate in the preparation of a kit or a diagnostic reagent.
  • Figure 1 is the reducing SDS-PAGE result of Anti-PLDH 10E5 antibody.
  • the present disclosure will be further described below in conjunction with specific examples, but the examples do not limit the present disclosure in any form.
  • the reagents, methods and equipment used in the present disclosure are conventional reagents, methods and equipment in the art.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof comprising the following CDRs:
  • Heavy chain CDR1 which comprises the amino acid sequence S-Y-T-M-H shown in SEQ ID NO.1, or consists of it;
  • a heavy chain CDR2 comprising or consisting of the amino acid sequence shown as H-I-N-P-S-S-G-Y-X1-N-X2-N-Q-K-F-X3-D, wherein X1 is I or L; X2 is I or L; X3 is K or Q; and
  • Heavy chain CDR3 which comprises the amino acid sequence T-G-T-G shown in SEQ ID NO.3, or consists of it;
  • antibody or antigen-binding fragment further comprises:
  • Light chain CDR1 which comprises the amino acid sequence T-A-S-S-S-V-S-S-S-G-Y-L-Q shown in SEQ ID NO.4, or consists of it;
  • Light chain CDR2 which comprises the amino acid sequence T-T-S-N-L-A-S shown in SEQ ID NO.5, or consists of it;
  • Light chain CDR3 which comprises the amino acid sequence H-Q-Y-H-R-S-P shown in SEQ ID NO.6, or consists of it;
  • the antibody or antigen-binding fragment can comprise a heavy chain CDR2 comprising a combination of amino acid residue substitutions as set forth in any one of SEQ ID NO. 19 to SEQ ID NO. 25.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.
  • antigen-binding fragment is a substance comprising part or all of the CDRs of an antibody which lacks at least some of the amino acids present in the full-length chain but which is still capable of specifically binding to the antigen. Such fragments are biologically active in that they bind to the antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope.
  • Such fragments are selected from Fab (composed of complete light chain and Fd), Fv (composed of VH and VL), scFv (single chain antibody, VH and VL are connected by a linker peptide) or single domain antibody ( Consists of VH only).
  • Such fragments can be produced by recombinant nucleic acid techniques, or can be produced by enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • CDRs refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more or even all of the anti-antibody The region of major amino acid residues that contribute to the binding affinity of an antigen-binding fragment thereof to the antigen or epitope it recognizes.
  • CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.
  • the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.
  • CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions.
  • framework region or "FR” region includes the heavy chain framework region and the light chain framework region, and refers to the region except the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein, heavy Chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising HFR1, HFR2, HFR3, and HFR4 framework regions; light chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising HFR1 , HFR2, HFR3 and HFR4 framework regions.
  • the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs Arrange and connect as follows: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • the antibody further comprises framework regions HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region, and framework regions LFR1, LFR2, LFR3, and LFR4 of the light chain variable region, wherein:
  • HFR1 comprises an amino acid sequence selected from SEQ ID NO:7 or having more than 90% homology with SEQ ID NO:7;
  • HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having more than 90% homology with SEQ ID NO: 8;
  • HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having more than 90% homology with SEQ ID NO: 9;
  • HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having more than 90% homology with SEQ ID NO: 10;
  • LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having more than 90% homology with SEQ ID NO: 11;
  • LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having more than 90% homology with SEQ ID NO: 12;
  • LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having more than 90% homology with SEQ ID NO: 13;
  • LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having more than 90% homology with SEQ ID NO: 14.
  • the HFR1 consists of SEQ ID NO: 7 or an amino acid sequence having more than 90% homology with SEQ ID NO: 7;
  • HFR2 consists of SEQ ID NO: 8 or an amino acid sequence having more than 90% homology with SEQ ID NO: 8;
  • HFR3 consists of SEQ ID NO: 9 or an amino acid sequence having more than 90% homology with SEQ ID NO: 9;
  • HFR4 consists of SEQ ID NO: 10 or an amino acid sequence having more than 90% homology with SEQ ID NO: 10;
  • LFR1 consists of SEQ ID NO: 11 or an amino acid sequence having more than 90% homology with SEQ ID NO: 11;
  • LFR2 consists of SEQ ID NO: 12 or an amino acid sequence having more than 90% homology with SEQ ID NO: 12;
  • LFR3 consists of SEQ ID NO: 13 or an amino acid sequence having more than 90% homology with SEQ ID NO: 13;
  • LFR4 consists of SEQ ID NO: 14 or an amino acid sequence having more than 90% homology with SEQ ID NO: 14.
  • the antibody further comprises a heavy chain variable region and a light chain variable region: the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 15, or consists of it; The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 16, or consists of it.
  • the antibody further comprises a heavy chain constant region and a light chain constant region;
  • the heavy chain constant region is any one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM species, the light chain constant region is a ⁇ chain or a ⁇ chain.
  • the species sources of the heavy chain constant region and the light chain constant region are bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, Deer, mink, chicken, duck, goose, turkey, gamecock or human.
  • the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 17, or consists of it; the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 18, or it consists of composition.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or consists of it.
  • the antibody or antigen-binding fragment comprises a heavy chain and a light chain, the light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 18.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , scFv, Fv, Fd, single chain antibody, diabody or domain antibody.
  • the present disclosure also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof.
  • Nucleic acids are usually RNA or DNA, and nucleic acid molecules can be single- or double-stranded.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA nucleic acid is used when it is ligated into a vector.
  • the present disclosure also relates to vectors containing said nucleic acids.
  • the present disclosure also relates to cells containing said nucleic acid or said vector.
  • the present disclosure also relates to an antibody conjugate comprising the antibody or an antigen-binding fragment thereof and a conjugation moiety conjugated thereto;
  • the coupling moiety includes a label selected from purification tags (such as His tags), detectable labels, such as colloidal gold, radioactive labels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels, electronic Dense labels such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme , carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labeling.
  • purification tags such as His tags
  • detectable labels such as colloidal gold, radioactive labels, luminescent substances, colored substances
  • enzymes such as fluorescent labels, chromophore labels, electronic Dense labels such as radioisotop
  • the present disclosure also relates to a kit or diagnostic reagent comprising said antibody or antigen-binding fragment or said antibody conjugate, wherein:
  • the kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to a Plasmodium antigen other than PLDH, including, for example, Plasmodium falciparum Plasmodium, P. vivax, P. malariae and/or P. ovale specific or consensus antigens, e.g. aldolase, e.g. HRP-II, e.g. Plasmodium antigens LSA-1, LSA-3, LSA- 5. SALSA, STARP, TRAP, PfEXP1, CS, MSP-3-1, MSP-3-2, MSP-3-5, MSP-3-6, MSP1, MSP4, MSP5, AMA-1, SERP and GLURP.
  • a Plasmodium antigen other than PLDH including, for example, Plasmodium falciparum Plasmodium, P. vivax, P. malariae and/or P. ovale specific or consensus antigens,
  • the kit or diagnostic reagent is for diagnosing malaria caused by Plasmodium.
  • the malaria is malaria caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and/or Plasmodium ovale.
  • the present disclosure also relates to methods of diagnosing malaria caused by Plasmodium in a subject, comprising:
  • the presence of the immune complex therein is indicative of the malaria.
  • the malaria is malaria caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and/or Plasmodium ovale.
  • the present disclosure also relates to a method of detecting Plasmodium or Plasmodium lactate dehydrogenase antigen in a test sample comprising:
  • the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment.
  • the immune complex further includes a second antibody that binds to the Plasmodium or Plasmodium lactate dehydrogenase antigen.
  • restriction enzymes and Prime Star DNA polymerase were purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the mRNA was extracted from the hybridoma cell line secreting Anti-PLDH 10E5 monoclonal antibody prepared by the inventor's laboratory, and the DNA product was obtained by RT-PCR method, which was inserted into pMD- after adding A reaction with rTaq DNA polymerase.
  • DH5 ⁇ competent cells were transformed, and after colonies were grown, 4 heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were cloned and sent to a gene sequencing company for sequencing.
  • variable region of light chain (VL) gene sequence is 324bp, which belongs to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, heavy
  • VH variable region of heavy chain
  • pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed.
  • the expression vector has introduced multiple cloning restriction sites such as HindIII, BamHI, EcoRI, etc., and named it pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T
  • the VL and VH gene-specific primers of the Anti-PLDH 10E5 antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, and 0.73 KB Light Chain gene fragment and 1.40kb Heavy Chain gene fragment.
  • the Heavy Chain and Light Chain gene fragments were respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were connected to the 3.4A expression vector to the Heavy Chain gene. Recombinant expression plasmids for Chain and Light Chain.
  • Step 2 Dilute the plasmid prepared in step (2) to 40 ⁇ g/100 ⁇ L with ultrapure water, adjust the CHO cells to 1.43 ⁇ 10 7 cells/mL in a centrifuge tube, mix 100 ⁇ L of the above plasmid with 700 ⁇ L of cells, transfer to the electroporation cup, electroporation , count the next day; 25 ⁇ mol/L MSX 96-well pressurized culture for about 25 days.
  • the cells were first cultured in a 125mL shake flask with an inoculation volume of 30mL, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/mL.
  • the expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. After that, expand the culture every 72 hours. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.
  • Shake flask parameters rotational speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Fed-batch feeding Start feeding daily after culturing in the shake flask for 72 hours.
  • HyClone TM Cell Boost TM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day , has been supplemented until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day.
  • Affinity purification was performed with a proteinA affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g of foreign control antibody as a control.
  • the electropherogram is shown in Figure 1. Two bands were revealed after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain).
  • amino acid sequences of HCDR1-HCDR3 of the 10E5 antibody are shown in SEQ ID NO.1-3 respectively; the amino acid sequences of LCDR1-3 are shown in SEQ ID NO.4-6; The amino acid sequences of the heavy chain variable region, the light chain variable region, the heavy chain and the light chain are respectively shown in SEQ ID NO.15-18.
  • the purified antibody was diluted to 10 ⁇ g/mL with PBST, and the Pan-PLDH antigen was also serially diluted with PBST;
  • KD means the equilibrium dissociation constant, that is, affinity
  • Kon means the association rate constant
  • Kd means the dissociation rate constant
  • the Pan-PLDH antigen Dilute the Pan-PLDH antigen to 3 ⁇ g/mL in the coating solution (the main component is NaHCO 3 ), 100 ⁇ L per well, overnight at 4°C; the next day, wash twice with the washing solution (the main component is Na 2 HPO 4 +NaCl), and pat dry ;Add blocking solution (20%BSA+80%PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; Add diluted purified antibody and control antibody, 100 ⁇ L/well, 37°C, 30min; wash 5 times with washing solution , pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37°C, 30 min; wash with washing solution 5 times, pat dry; add 50 ⁇ L/well of chromogenic solution A (the main components are citric acid, sodium acetate, acetyl aniline and carbamide peroxide), add 50 ⁇ L/well of chromogenic solution B (mainly composed of citric acid, EDTA ⁇ 2Na
  • the monoclonal antibody prepared in Example 1 was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples were taken for state observation at 7 days, 14 days, and 21 days, and The activity test was carried out on the samples left for 21 days (the activity of the samples was assessed by the OD results of enzyme immunoassay).
  • Embodiment 4 performance evaluation
  • the antibodies prepared in the foregoing examples and the control antibody were used as labeled antibodies, and were used together with another strain of anti-Pan-PLDH antibody (purchased from Fipeng) as a coating antibody, and compared on the colloidal gold platform.
  • the difference between the prepared antibody and the control antibody in terms of color development in the immunoassay shows that the antibody prepared in the disclosed embodiment can achieve a better performance level than the control. See the table below for details:
  • the color development of gold standard is composed of C plus numbers. The smaller the number after C, the stronger the color development and the higher the activity; the higher the number after C, the weaker the color development and lower activity; the number is followed by "+” It is slightly stronger than without color by 0.5-1C, and the number with "-" is slightly lower than without color by 0.5-1C. B means no or very weak color development.
  • the present disclosure provides a PLDH monoclonal antibody, which can simultaneously detect Plasmodium falciparum, P. vivax, P. malariae and P. ovale, and has excellent antibody affinity, reactivity, specificity and sensitivity.

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Abstract

L'invention concerne un anticorps contre le plasmodium et son application. L'anticorps monoclonal de la protéine lactate déshydrogénase préparé contre le plasmodium peut détecter simultanément le plasmodium falciparum, le plasmodium vivax, le plasmodium falciparum et le plasmodium ovale, et présente des avantages évidents en termes d'affinité, d'activité de réaction, de spécificité et de sensibilité.
PCT/CN2022/115075 2021-09-10 2022-08-26 Anticorps contre le plasmodium et son application Ceased WO2023035974A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117947199A (zh) * 2024-03-26 2024-04-30 江苏硕世生物科技股份有限公司 一种用于区分疟原虫虫种的引物组合及试剂盒
CN118725109A (zh) * 2023-03-30 2024-10-01 东莞市朋志生物科技有限公司 抗acth抗体、检测acth的试剂和试剂盒
WO2025031261A1 (fr) * 2023-08-10 2025-02-13 菲鹏生物股份有限公司 Anticorps dirigé contre le plasmodium ou la lactate déshydrogénase de celui-ci et utilisation d'anticorps

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117467005A (zh) * 2023-11-15 2024-01-30 深圳重链生物科技有限公司 抗疟原虫单克隆抗体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024141A1 (fr) * 1995-12-29 1997-07-10 Piper Robert C Anticorps monoclonaux et methode de capture immunologique pour la quantification et la differenciation d'especes d'hematozoaires
KR20030088294A (ko) * 2002-05-14 2003-11-19 주식회사 에스디 신속 면역크로마토그라피법 및 효소면역측정법에 의해사람 전혈에서 말라리아 항원 젖산탈수소효소를 검출하는방법 및 진단 키트
CN102590522A (zh) * 2012-03-07 2012-07-18 上海凯创生物技术有限公司 疟原虫抗体胶体金检测试剂盒及其制备
CN110146697A (zh) * 2019-06-03 2019-08-20 中国疾病预防控制中心寄生虫病预防控制所 一种快速检测恶性疟、间日疟、三日疟和卵形疟的免疫试纸条

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363044B (zh) * 2018-12-25 2021-10-12 东莞市朋志生物科技有限公司 一种抗泛种特异性疟原虫乳酸脱氢酶的抗体
CN110951703B (zh) * 2019-12-23 2023-04-07 杭州贤至生物科技有限公司 一种间日疟原虫乳酸脱氢酶重组蛋白及其单克隆抗体的制备

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024141A1 (fr) * 1995-12-29 1997-07-10 Piper Robert C Anticorps monoclonaux et methode de capture immunologique pour la quantification et la differenciation d'especes d'hematozoaires
KR20030088294A (ko) * 2002-05-14 2003-11-19 주식회사 에스디 신속 면역크로마토그라피법 및 효소면역측정법에 의해사람 전혈에서 말라리아 항원 젖산탈수소효소를 검출하는방법 및 진단 키트
CN102590522A (zh) * 2012-03-07 2012-07-18 上海凯创生物技术有限公司 疟原虫抗体胶体金检测试剂盒及其制备
CN110146697A (zh) * 2019-06-03 2019-08-20 中国疾病预防控制中心寄生虫病预防控制所 一种快速检测恶性疟、间日疟、三日疟和卵形疟的免疫试纸条

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GALATAS BEATRIZ, MAYOR ALFREDO, GUPTA HIMANSHU, BALANZA NÚRIA, JANG IHN KYUNG, NHAMUSSUA LIDIA, SIMONE WILSON, CISTERÓ PAU, CHIDIM: "Field performance of ultrasensitive and conventional malaria rapid diagnostic tests in southern Mozambique", MALARIA JOURNAL, vol. 19, no. 1, 1 December 2020 (2020-12-01), XP093045859, DOI: 10.1186/s12936-020-03526-9 *
KANAYO IFEORAH IFEANYI, BROWN BIOBELE J, SODEINDE OLUGBEMIRO O: "A COMPARISON OF RAPID DIAGNOSTIC TESTING (BY PLASMODIUM LACTATE DEHYDROGENASE), AND QUANTITATIVE BUFFY COAT TECHNIQUE IN MALARIA DIAGNOSIS IN CHILDREN", AFRICAN JOURNAL OF INFECTIOUS DISEASES, vol. 11, no. 2, 8 July 2017 (2017-07-08), pages 31 - 38, XP093045862, ISSN: 2006-0165, DOI: 10.21010/ajid.v11i2.5 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118725109A (zh) * 2023-03-30 2024-10-01 东莞市朋志生物科技有限公司 抗acth抗体、检测acth的试剂和试剂盒
WO2025031261A1 (fr) * 2023-08-10 2025-02-13 菲鹏生物股份有限公司 Anticorps dirigé contre le plasmodium ou la lactate déshydrogénase de celui-ci et utilisation d'anticorps
CN117947199A (zh) * 2024-03-26 2024-04-30 江苏硕世生物科技股份有限公司 一种用于区分疟原虫虫种的引物组合及试剂盒

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