CN115785275B

CN115785275B - Anti-plasmodium antibody and application thereof

Info

Publication number: CN115785275B
Application number: CN202111059915.6A
Authority: CN
Inventors: 孟媛; 钟冬梅; 周全兴; 黄玉玲; 熊俊文
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2023-09-22
Anticipated expiration: 2041-09-10
Also published as: CN115785275A; WO2023035974A1

Abstract

The invention relates to an anti-plasmodium antibody and application thereof. The anti-plasmodium lactic dehydrogenase protein monoclonal antibody prepared by the invention can detect plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale simultaneously, has obvious advantages in the aspects of affinity, reactivity, specificity and sensitivity compared with the existing main stream raw materials, and has great market potential.

Description

Anti-plasmodium antibody and application thereof

Technical Field

The invention belongs to the technical field of antibodies. More particularly, to an anti-plasmodium antibody and uses thereof.

Background

Malaria (Malaria) is an insect-borne infectious disease caused by infection of Malaria parasites by biting or infusing blood of plasmodium-carrying persons, and is classified by the world health organization (World Health Organization, WHO) as a global three-major public health problem together with AIDS and tuberculosis. According to WHO, world malaria report 2019, 2.28 million cases of malaria co-occur worldwide in 2018, 40.5 tens of thousands of people die from the disease. There are four major plasmodium species that are parasitic to the human body: plasmodium falciparum (Plasmodium falciparum, pf), plasmodium vivax (pv), plasmodium malariae (Plasmodium malariae, pm), plasmodium ovale (po). Of the plasmodium infested with humans, plasmodium falciparum is the most pathogenic.

After the plasmodium bites the human body by the female anopheles, sporozoites firstly invade liver cells and propagate and develop in the liver cells, and when the liver cells are broken, a large amount of released merozoites quickly enter the blood circulation to invade red blood cells; not only does the massive destruction of erythrocytes cause anemia during the asexual reproduction in erythrocytes, but also many metabolites are released as pyrogens, stimulating the body to produce a strong protective immune response. The clinical manifestations of malaria vary according to the course of the disease, including fever, chills, and headache, and may also manifest as more severe symptoms, including severe anemia, respiratory distress associated with metabolic acidosis, multiple organ failure, and cerebral malaria, with these severe changes collectively referred to as severe malaria.

The detection technology is classified in principle, and the current method for detecting plasmodium is divided into four types. Firstly, directly detecting plasmodium by a microscope, which is the gold standard for malaria diagnosis in clinic at present, but the method is time-consuming and labor-consuming, and requires skilled technicians and certain experimental conditions. Secondly, the method has high sensitivity and specificity, but needs more complicated 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 in a basic layer. Thirdly, the method needs a professional detection instrument for detecting the hemozoin, is generally used for laboratory research, and is not suitable for field detection. Fourthly, the method for detecting the plasmodium through the immune response of the antigen and the antibody is simple to operate, high in speed and high in accuracy, has low requirements on laboratories and operators, is widely suitable for basic level screening of clinical laboratory and disease control system laboratories and the like, and has very important functions on initial detection of malaria, successful control of outbreak and guiding treatment in hospitals and communities. Rapid immunodiagnostic products based on detection of plasmodium-specific antigens are therefore increasingly gaining importance in malaria diagnosis.

Commonly used detection malaria antigens are mainly Histidine-rich protein II (HRP-II) specific to Plasmodium falciparum and Plasmodium lactate dehydrogenase (Plasmodium lactate dehydrogenase, PLDH). HRP-II is the most accepted diagnostic marker of plasmodium falciparum, but the detection kit of plasmodium falciparum with HRP-II as target antigen can only be used for detecting plasmodium falciparum, and cannot be detected for plasmodium falciparum with wider popularity; when the living insect bodies in the blood of a patient disappear, the HRP-II still exists in the human body for a long time, and the infection period cannot be accurately confirmed; cross-reacting an anti-HRP-II monoclonal antibody with a rheumatoid factor causes a pseudo-positive reaction. The plasmodium lactate dehydrogenase is an important enzyme for ensuring normal glycolysis of plasmodium, has significantly different physical and biochemical characteristics compared with human erythrocytes and other lactate dehydrogenase of a plurality of micro-substances, is a protein which is necessary to be expressed in the process of life activities of plasmodium, has higher abundance, and therefore becomes an important target for detecting plasmodium. Since PLDH is produced only from living malaria parasites, the method of using PLDH as a detection antigen can also identify the dead or alive of the insect bodies in a patient, and thus can evaluate the monitoring of the therapeutic effect and the condition of post-combustion. In addition, the four plasmodium-produced PLDH have different isoforms, with antigens specific to species and genus, and can be largely divided into two major classes: firstly, PLDH with species specificity comprises pf PLDH, pv PLDH, pm PLDH and po PLDH, and monoclonal antibodies produced by taking the PLDH as target proteins only recognize PLDH of specific plasmodium species; secondly, the general species specific antigen plasmodium lactate dehydrogenase (Pan-PLDH) is used as a monoclonal antibody produced by target proteins to recognize PLDH of four plasmodium species.

At present, the raw materials of the Pan-PLDH monoclonal antibodies capable of simultaneously detecting the plasmodium falciparum, the plasmodium vivax, the plasmodium malariae and the plasmodium ovale are less, mainly depends on import, and has certain defects of specificity and sensitivity.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defect of the specificity and sensitivity of the prior Pan-PLDH monoclonal antibody for simultaneously detecting falciparum malaria, vivax malaria, malarial of three days and oval malaria; the PLDH monoclonal antibody prepared by the invention can detect plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale simultaneously, and has obvious advantages in the aspects of antibody affinity, reactivity, specificity and sensitivity compared with the existing main stream raw materials.

It is an object of the present invention to provide an antibody or antigen binding fragment comprising the following CDRs:

a heavy chain CDR1 comprising or consisting of the amino acid sequence S-Y-T-M-H shown in SEQ ID NO. 1;

a heavy chain CDR2 comprising or consisting of an amino acid sequence depicted by H-I-N-P-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

a heavy chain CDR3 comprising or consisting of the amino acid sequence T-G-T-G shown in SEQ ID No. 3;

and the antibody or antigen binding fragment further comprises:

a light chain CDR1 comprising or consisting of the amino acid sequence T-A-S-S-S-V-S-S-G-Y-L-Q shown in SEQ ID NO. 4;

a light chain CDR2 comprising or consisting of the amino acid sequence T-S-N-L-a-S shown in SEQ ID No.5 And

a light chain CDR3 comprising or consisting of the amino acid sequence H-Q-Y-H-R-S-P shown in SEQ ID No. 6.

It is another object of the invention to provide nucleic acids, vectors or cells related to said antibodies or antigen binding fragments.

The invention also provides methods of making the antibodies or antigen binding fragments.

The invention also provides an antibody conjugate and a kit/diagnostic reagent comprising the antibody or antigen binding fragment or the antibody conjugate.

The invention also provides the use of the antibody or antigen binding fragment or the antibody conjugate in the preparation of a kit or diagnostic reagent.

Drawings

FIG. 1 shows the result of reducing SDS-PAGE of Anti-PLDH 10E5 antibody.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The present invention relates to an antibody or antigen binding fragment thereof comprising the following CDRs:

and the antibody or antigen binding fragment further comprises:

a light chain CDR2 comprising or consisting of the amino acid sequence T-S-N-L-a-S shown in SEQ ID No.5 And a light chain CDR3 comprising or consisting of the amino acid sequence H-Q-Y-H-R-S-P shown in SEQ ID NO. 6;

for example, the antibody or antigen binding fragment may comprise a heavy chain CDR2 comprising the amino acid residue substitution combination shown in any one of SEQ ID No.19 to SEQ ID No. 25.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity. The term "antigen binding fragment" is a substance comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab (consisting of intact light chain and Fd), fv (consisting of VH and VL), scFv (single chain antibody, with a linker peptide between VH and VL) or single domain antibody (consisting of VH only). Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues contributing to the binding affinity of an antibody or antigen binding fragment thereof to an antigen or epitope recognized by the antibody or antigen binding fragment thereof. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, and the 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In some embodiments, 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 to 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; and

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.

In some embodiments, 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; and

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.

In some embodiments, 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 shown as SEQ ID NO.15 or consists of the same; the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 16 or consists of the same.

In some embodiments, 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, and the light chain constant region is a kappa chain or a lambda chain.

In some embodiments, the species source of the heavy and light chain constant regions is cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock, or human.

In some embodiments, the heavy chain of the antibody has or consists of the amino acid sequence shown in SEQ ID NO. 17; the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 18 or consists of the same.

In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') ₂ scFv, fv, fd, single chain antibody, diabody or domain antibody.

The invention also relates to nucleic acids encoding the antibodies or antigen binding fragments thereof.

Nucleic acids are typically RNA or DNA, and nucleic acid molecules may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are used when they are incorporated into vectors.

The invention also relates to a vector containing said nucleic acid.

The invention also relates to a cell containing said nucleic acid or said vector.

The invention also relates to an antibody conjugate comprising the antibody or antigen-binding fragment thereof and a coupling moiety coupled thereto;

alternatively, the coupling moiety comprises a label selected from the group consisting of a purification tag (e.g. His tag), 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, luciferin, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin-labels.

The invention also relates to a kit or diagnostic reagent comprising said antibody or antigen binding fragment or said antibody conjugate, wherein:

optionally, the kit or diagnostic reagent further comprises antibodies or antigen binding fragments, antibody conjugates, or fusion proteins thereof that bind to plasmodium antigens other than PLDH, including, for example, plasmodium falciparum, plasmodium vivax, plasmodium malariae and/or plasmodium ovale specific antigens or consensus antigens, such as aldolases, 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.

The use of said antibody or antigen binding fragment thereof or said antibody conjugate in the preparation of a kit or diagnostic reagent is also within the scope of the present invention.

Embodiments of the present invention will be described in detail below with reference to examples.

In the following examples, restriction enzymes, prime Star DNA polymerase were purchased from Takara corporation. pMD-18T vector was purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMARTTMRACE cDNA Amplification Kit kit was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.

Example 1 preparation of anti-the pan-specific antigen plasmodium lactate dehydrogenase antibody (10E 5 antibody)

1. Construction of expression plasmid

(1) Preparation of 10E5 antibody Gene

mRNA is extracted from hybridoma cell strain secreting Anti-PLDH 10E5 monoclonal antibody, DNA product is obtained through RT-PCR method, the product is inserted into pMD-18T vector after A adding reaction by rTaq DNA polymerase, DH5 alpha competent cells are transformed, after colony is grown, heavy Chain (heavychain) and Light Chain (Light Chain) gene clone are respectively taken for sequencing by 4 clone gene sequencing companies.

(2) Sequence analysis of 10E5 antibody variable region Gene

The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct; the Light Chain variable region (variable region of Light Chain, VL) gene sequence in the gene fragment amplified by the Light Chain belongs to the VkII gene family, and a 57bp leader peptide sequence is arranged in front of the Light Chain variable region; in the gene fragment amplified by the heavychain primer pair, the Heavy Chain variable region (variable region of Heavy Chain, VH) gene sequence is 348bp, belongs to the VH1 gene family, and has a 57bp leader peptide sequence in front of the Heavy Chain variable region.

(3) Construction of recombinant antibody expression plasmids

pcDNATM3.4vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, named pcDNA3.4A expression vector and is hereinafter abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in the pMD-18T vector, VL and VH gene specific primers of the Anti-PLDH 10E5 antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a Light Chain gene fragment of 0.73KB and a Heavy Chain gene fragment of 1.40KB are amplified by a PCR amplification method.

The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene and the Light Chain gene after the fragment and the vector are purified and recovered are connected into the 3.4A expression vector to form recombinant expression plasmids of the Heavy Chain and the Light Chain.

2. Stable cell line selection

(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity

Diluting the plasmid prepared in step 1- (3) with ultrapure water to 40. Mu.g/100. Mu.L, and regulating CHO cells to 1.43X10 ⁷ 100. Mu.L of the plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sampled and counted on days 3, 5 and 7, and collected and detected on day 7.

Coating liquid (NaHCO as main component) ₃ ) PAN-PLDH antigen (from Figpeng organism) was diluted to 3. Mu.g/mL, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing liquid (main component is Na ₂ HPO ₄ +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding 50 μL/Kong Xianse solution A (mainly comprising citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding 50 μL/Kong Xianse solution B (mainly comprising citric acid, EDTA.2Na, TMB and concentrated HCl) for 10min; adding stop solution (EDTA.2Na and concentrated H as main components) ₂ SO ₄ ) 50. Mu.L/well; OD was read on the microplate reader at 450nm (reference 630 nm).

The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that the antibodies produced after transient plasmid transformation were active against the Pan-PLDH antigen.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50. Mu.L of Buffer, 100. Mu.g/tube of plasmid prepared in step 1- (3), 10. Mu.L of PvuI enzyme, 500. Mu.L of sterile water, and performing water bath enzyme digestion at 37 ℃ overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain

The plasmid prepared in step 2- (2) was diluted to 40. Mu.g/100. Mu.L with ultrapure water, and CHO cells were regulated to 1.43X10 ⁷ Placing cells/mL in a centrifuge tube, mixing 100 μl of the plasmid with 700 μl of cells, transferring into an electrorotating cup, electrorotating, and counting the next day; 25 mu mol/L MSX 96 wells were incubated under pressure for approximately 25 days.

Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 ⁶ Batch culture was performed with cells/mL and 2.2mL, and cell density was 0.3X10 ⁶ Performing seed preservation by using cells/mL and 2 mL; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.

3. Recombinant antibody production

(1) Cell expansion culture

After cell recovery, the cells were first cultured in 125mL shake flasks with an inoculation volume of 30mL and a medium of 100% Dynamis, and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/mL, and calculating the expanded culture volume according to the production requirement, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/mL for production.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding, hyClone, was started daily when cultured in shake flasks for 72h ^TM Cell Boost ^TM Feed 7a fed dailyFeed 7b was fed daily at 3% of the initial culture volume, one thousandth of the initial culture volume, up to day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 4. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of external control antibody was used as a control, and the electrophoresis pattern was shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, one Mr was 50KD (heavy chain) and the other was 28KD (light chain).

Sequencing the purified antibody and Kabat analyzing to obtain the amino acid sequences of HCDR1-HCDR3 of the 10E5 antibody as shown in SEQ ID NO. 1-3; the amino acid sequence of LCDR1-3 is shown in SEQ ID NO. 4-6; the heavy chain variable region, the light chain variable region, the heavy chain and the light chain of the antibody have amino acid sequences shown in SEQ ID NO.15-18 respectively.

A series of single point mutations was performed on 10E5, steps 1- (3) to 3- (2) were repeated, and 10E5RMb1 to 10E5RMb7 were obtained by screening. The amino acid sequences of the 10E5RMb1 to 10E5RMb7 and 10E5 antibodies were only different from each other in HCDR2, and the HCDR2 of the 10E5RMb1 to 10E5RMb7 were shown in SEQ ID NO.19-25, respectively, as determined by sequencing and Kabat analysis.

Example 2 affinity assay and Activity characterization

1. Affinity analysis

The purified antibody was diluted to 10. Mu.g/mL with PBST using an AMC sensor, and the Pan-PLDH antigen was also subjected to gradient dilution with PBST;

the operation flow is as follows: buffer 1 (PBST; main component is Na) ₂ HPO ₄ Equilibration for 60s, naCl and Tween 20), antibody 300s immobilized in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM GLY solution pH1.69 and buffer 3, and data output.

TABLE 1

Note that: KD represents equilibrium dissociation constant, i.e. affinity; kon represents the binding rate constant; kdis represents the dissociation rate constant.

2. Activity assay

Coating liquid (NaHCO as main component) ₃ ) The Pan-PLDH antigen was diluted to 3. Mu.g/mL, 100. Mu.L per well, overnight at 4 ℃; the next day, the washing liquid (main component is Na ₂ HPO ₄ +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted purified antibody and control antibody, 100 μl/well, 37deg.C, 30min; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding 50 μL/Kong Xianse solution A (mainly comprising citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding 50 μL/Kong Xianse solution B (mainly comprising citric acid, EDTA.2Na, TMB and concentrated HCl) for 10min; adding stop solution (EDTA.2Na and concentrated H as main components) ₂ SO ₄ ) 50. Mu.L/well; OD was read on the microplate reader at 450nm (reference 630 nm).

TABLE 2

Sample concentration (ng/mL)	25.000	12.500	6.250	3.125	1.563	0.000
							Control	1.276	0.629	0.487	0.147	0.072	0.048
10E5	1.431	0.867	0.624	0.265	0.111	0.038
							10E5RMb1	1.237	0.699	0.437	0.177	0.032	0.048
10E5RMb2	1.352	0.724	0.542	0.199	0.089	0.044
							10E5RMb3	1.512	0.884	0.613	0.243	0.123	0.051
10E5RMb4	1.414	0.781	0.596	0.212	0.095	0.046
							10E5RMb5	1.393	0.797	0.574	0.209	0.091	0.045
10E5RMb6	1.451	0.825	0.611	0.235	0.109	0.049
							10E5RMb7	1.428	0.817	0.609	0.241	0.112	0.047

OD values in Table 2 indicate that Anti-PLDH 10E5 and 10E5RMb1-7 monoclonal antibodies were active well.

Example 3 stability assessment

The monoclonal antibody prepared in example 1 was placed at 4 ℃, -80 ℃, -37 ℃ (refrigerator) for 21 days, samples were taken for 7 days, 14 days and 21 days for status observation, and activity detection was performed on the samples placed for 21 days (activity of the samples was checked using the results of enzyme-free detection OD).

The stability test results of the 10E5 antibody are shown in Table 3, and the results show that the 10E5 and 10E5RMb1-7 single gram antibodies do not have obvious protein state change after being placed for 21 days under three examination conditions, and the activity of the single gram antibodies does not have a descending trend along with the increase of the examination temperature, so that the stability of the anti-pan-species specific antigen plasmodium lactate dehydrogenase antibody prepared by the invention is good.

TABLE 3 Table 3

Sample concentration (ng/mL)	25	12.5	0
				4 ℃,21 days sample	1.314	0.808	0.094
Sample at-80℃for 21 days	1.299	0.838	0.083
				37 ℃ and 21 days of sample	1.305	0.896	0.067

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Dongguan City, pengzhi biotechnology Co., ltd

<120> an anti-plasmodium antibody and use thereof

<130> P2021036CN01

<160> 25

<170> PatentIn version 3.5

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<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 4

Thr Ala Ser Ser Ser Val Ser Ser Gly Tyr Leu Gln

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 5

Thr Thr Ser Asn Leu Ala Ser

1 5

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 6

His Gln Tyr His Arg Ser Pro

1 5

<210> 7

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

20 25 30

<210> 8

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 8

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

1 5 10

<210> 9

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 9

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

1 5 10 15

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

20 25 30

<210> 10

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 10

Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

<210> 11

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 11

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys

20

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 12

Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ile Trp Ile Tyr

1 5 10 15

<210> 13

<211> 32

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 13

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

1 5 10 15

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

<210> 14

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 14

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 15

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 15

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly His Ile Asn Pro Ser Ser Gly Tyr Leu Asn Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Thr Gly Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 16

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 16

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Gln Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ile Trp

35 40 45

Ile Tyr Thr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro

85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 440

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 17

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly His Ile Asn Pro Ser Ser Gly Tyr Leu Asn Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Thr Gly Thr Gly Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala

115 120 125

Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu

130 135 140

Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly

145 150 155 160

Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp

165 170 175

Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro

180 185 190

Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys

195 200 205

Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile

210 215 220

Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val

245 250 255

Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val

260 265 270

Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala

305 310 315 320

Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro

325 330 335

Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala

340 345 350

Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu

355 360 365

Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr

370 375 380

Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr

385 390 395 400

Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe

405 410 415

Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys

420 425 430

Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 18

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 18

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Gln Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ile Trp

35 40 45

Ile Tyr Thr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro

85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala

100 105 110

Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser

115 120 125

Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp

130 135 140

Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val

145 150 155 160

Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met

165 170 175

Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser

180 185 190

Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys

195 200 205

Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 19

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 19

His Ile Asn Pro Ser Ser Gly Tyr Ile Asn Ile Asn Gln Lys Phe Gln

1 5 10 15

Asp

<210> 20

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 20

His Ile Asn Pro Ser Ser Gly Tyr Ile Asn Leu Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 21

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 21

His Ile Asn Pro Ser Ser Gly Tyr Leu Asn Ile Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 22

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 22

His Ile Asn Pro Ser Ser Gly Tyr Ile Asn Leu Asn Gln Lys Phe Gln

1 5 10 15

Asp

<210> 23

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 23

His Ile Asn Pro Ser Ser Gly Tyr Leu Asn Ile Asn Gln Lys Phe Gln

1 5 10 15

Asp

<210> 24

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 24

His Ile Asn Pro Ser Ser Gly Tyr Ile Asn Ile Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 25

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 25

His Ile Asn Pro Ser Ser Gly Tyr Leu Asn Leu Asn Gln Lys Phe Gln

1 5 10 15

Asp

Claims

1. An antibody or antigen-binding fragment comprising the following CDRs: the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO. 1; a heavy chain CDR2 having the amino acid sequence 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 a heavy chain CDR3, the amino acid sequence of which is shown in SEQ ID NO. 3; and the antibody or antigen binding fragment further comprises: light chain CDR1 with amino acid sequence shown in SEQ ID No. 4; light chain CDR2 with amino acid sequence shown in SEQ ID No. 5; and a light chain CDR3, the amino acid sequence of which is shown in SEQ ID NO. 6.

2. The antibody or antigen-binding fragment of claim 1, further comprising 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 is selected from SEQ ID NO 7 or an amino acid sequence having more than 90% homology with SEQ ID NO 7; HFR2 is selected from SEQ ID NO 8 or an amino acid sequence having more than 90% homology with SEQ ID NO 8; HFR3 is selected from SEQ ID NO 9 or an amino acid sequence having more than 90% homology with SEQ ID NO 9; HFR4 is selected from SEQ ID NO 10 or an amino acid sequence having more than 90% homology with SEQ ID NO 10; and LFR1 is selected from SEQ ID NO. 11 or an amino acid sequence having more than 90% homology with SEQ ID NO. 11; LFR2 is selected from SEQ ID NO. 12 or an amino acid sequence having more than 90% homology with SEQ ID NO. 12; LFR3 is selected from SEQ ID NO. 13 or an amino acid sequence having more than 90% homology with SEQ ID NO. 13; LFR4 is selected from SEQ ID NO. 14 or an amino acid sequence having more than 90% homology with SEQ ID NO. 14.

3. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 15; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

4. The antibody or antigen binding fragment of claim 1, further comprising a heavy chain constant region selected from the group consisting of heavy chain constant regions of any one or more of IgG1, igG2, igG3, igG4, igA, igD, igE, or IgM; the light chain constant region is a kappa chain or a lambda chain.

5. The antibody or antigen binding fragment of claim 4, wherein the species source of the heavy and light chain constant regions is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, nugget, or human.

6. The antibody or antigen binding fragment of claim 4, wherein the species source of the heavy and light chain constant regions is dairy cows.

7. The antibody or antigen binding fragment of claim 1, which is selected from Fab, fab ', F (ab') 2, scfv, fv, fd, diabody, or domain antibody.

8. A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 7.

9. A cell comprising the nucleic acid of claim 8.

10. A method of producing the antibody or antigen-binding fragment of any one of claims 1 to 7, comprising culturing the cell of claim 9.

11. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7 and a conjugate moiety conjugated thereto.

12. The antibody conjugate of claim 11, wherein the conjugate moiety is selected from the group consisting of a purification tag and a detectable label.

13. The antibody conjugate of claim 12, wherein the detectable label is selected from the group consisting of colloidal gold, a radiolabel, a luminescent material, a colored material, an enzyme.

14. The antibody conjugate of claim 12, wherein the detectable label is selected from the group consisting of fluorescent labels, chromophore labels, electron dense labels.

15. The antibody conjugate of claim 12, wherein the detectable label is selected from one or more of radioisotope, 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, spin-labeling.

16. A kit or diagnostic reagent comprising the antibody or antigen-binding fragment of any one of claims 1 to 7 or the antibody conjugate of any one of claims 11 to 15.

17. The kit or diagnostic reagent of claim 16, further comprising an antibody or antigen binding fragment, antibody conjugate, or fusion protein that binds to a plasmodium antigen other than plasmodium lactate dehydrogenase protein, including plasmodium falciparum, plasmodium vivax, plasmodium malariae, and/or plasmodium ovale specific antigen or consensus antigen.

18. The kit or diagnostic reagent of claim 17, wherein the plasmodium antigen other than plasmodium lactate dehydrogenase protein is selected from HRP-II or aldolase.

19. The kit or diagnostic reagent of claim 17, wherein the plasmodium antigen other than plasmodium lactate dehydrogenase protein is selected from 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.

20. Use of an antibody or antigen binding fragment according to any one of claims 1 to 7 or an antibody conjugate according to any one of claims 11 to 15 in the preparation of a kit or diagnostic reagent for detecting plasmodium.