WO2009075404A1 - Trousse de diagnostic d'une infection mixte de malaria par le plasmodium vivax et le plasmodium falciparum utilisant des anticorps spécifiques de la lactate déshydrogénase du plasmodium vivax et du plasmodium falciparum - Google Patents

Trousse de diagnostic d'une infection mixte de malaria par le plasmodium vivax et le plasmodium falciparum utilisant des anticorps spécifiques de la lactate déshydrogénase du plasmodium vivax et du plasmodium falciparum Download PDF

Info

Publication number
WO2009075404A1
WO2009075404A1 PCT/KR2007/006962 KR2007006962W WO2009075404A1 WO 2009075404 A1 WO2009075404 A1 WO 2009075404A1 KR 2007006962 W KR2007006962 W KR 2007006962W WO 2009075404 A1 WO2009075404 A1 WO 2009075404A1
Authority
WO
WIPO (PCT)
Prior art keywords
vivax
falciparum
kit
plasmodium
monoclonal antibody
Prior art date
Application number
PCT/KR2007/006962
Other languages
English (en)
Inventor
Chom Kyu Chong
Yoon Kong
Jong Jin Park
Kwang Myun Cheong
Jai Cheol Yoo
Sang Oh Lee
Ha Na Oh
Sun Ock Park
Original Assignee
Bioland Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bioland Ltd. filed Critical Bioland Ltd.
Publication of WO2009075404A1 publication Critical patent/WO2009075404A1/fr

Links

Classifications

    • 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
    • G01N33/56905—Protozoa
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/445—Plasmodium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • 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
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to monoclonal antibodies specific to Plasmodium vivax lactate dehydrogenase and Plasmodium falciparum lactate dehydrogenase, respectively, and a kit for diagnosing malaria comprising both of them, and a method of diagnosing malaria using the same. More particularly, the present invention relates to the world's first technique for diagnosis of mixed malaria infection. Further, the present invention also relates to a monoclonal antibody used for the kit and hybridoma for producing the same.
  • Malaria is an infectious disease carried by a mosquito. Annually, three to five hundred million people are infected with malaria throughout the world, of which about 2.7 million people die due to infection.
  • Plasmodium parasite that can infect humans : P lasmodium falciparum (P. falciparum) , Plasmodium vivax (P. vivax) , Plasmodium ovale (P. ovale) , and Plasmodium malariae (P. malariae) , in which P. falciparum is the most serious and causes high fatality, and P. vivax is the most widely spread species.
  • malaria also called miasmatic fever
  • malaria was reported to have been completely exterminated in the early 1980s by the efforts of the Korean government and WHO.
  • malaria since malaria re-emerged in 1993 by mosquitoes migrating from North Korea to South Korea across the DMZ, the number of malaria infections is continuously increasing.
  • travelers infected with Plasmodium falciparum in addition to indigenous Plasmodium vivax were treated in Korea.
  • the life cycle of malaria parasites is categorized into two stages: asexual reproduction in which malaria parasites develop in humans and sexual reproduction in which the malaria parasites develop in mosquitoes.
  • sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver. Then, the sporozoites infect the liver cells and multiply into merozoites therein. The merozoites escape from the liver and enter red blood cells, where they multiply several hundred folds, and then rupture the red blood cells to escape back into the bloodstream. Some of the merozoites turn into gametocytes. If a mosquito pierces the skin of an infected person, it potentially picks up gametocytes within the blood. These gametocytes multiply in the mosquito's mid gut and transfer to the mosquito's salivary glands to turn into sporozoites, which are latent until the mosquito bites a person.
  • the blood smear analysis is a process in which thin blood smears prepared on a slide glass are stained and examined under a microscope to locate red blood cells infected with malaria.
  • This technique has advantages in that the cost of each test is considerably low; it is possible to diagnose malaria infection even if the blood includes only 5 to 10 malaria Plasmodia per ml; and it can distinguish four types of malaria Plasmodia.
  • an expensive device, such as a microscope is required; only an expert can correctly distinguish malaria infection; and it is not appropriate for mass tests.
  • PCR Polymerase chain reaction
  • HRP-2 histidine rich protein-2
  • Plasmodium lactate dehydrogenase was first separated from blood infected with malaria by D. L. Vander Jagt et al. in 1981. Makler reported in 1992 that malaria infection could be diagnosed using Plasmodium lactate dehydrogenase. Thereafter, in 1992, Piper et al. reported a kit in which P. falciparum lactate dehydrogenase genes are cloned to prepare monoclonal antibodies, which are used to distinguish between P. falciparum and P. vivax (Piper, R. C, et al., Am. J. Trop. Med. Hyg. 60 (1): 109-118, 1999). However, as P.
  • vivax lactate dehydrogenase gene generally found in Asia, was only cloned recently, monoclonal antibodies binding thereto have yet to be developed.
  • the development of monoclonal antibodies specific to P. vivax lactate dehydrogenase is achieved by the present invention.
  • the biochemical properties of Plasmodium aldolase were fist discovered by H. Dobeli et al . by preparing a recombinant protein from blood of a patient infected with P. falciparum and purifying the same. In 1999, Tjitra et al.
  • kits consisting of monoclonal antibodies against Plasmodium aldolase and HRP-2 based on a rapid immunochromatography, and employed the kit to diagnosis malaria infection (Tjitra et al. (1999) J. Clin. Microbiol. 37, 2412-2417). They used monoclonal antibodies against HRP-2 for diagnosis of P. falciparum, and monoclonal antibodies against aldolase for diagnosis of P. vivax.
  • the kit has specificity and sensitivity of 94% and 98%, respectively, but was reported to remarkably decrease in positivity rate if malaria Plasmodia density was 500 or less per ⁇ l of blood. HRP-2 is detected only in the case of infection with P. falciparum, so that infection with P.
  • the present invention has been conceived to solve the problems of the conventional techniques as described above, and the inventors of the present invention have developed a kit for diagnosis of malaria, which exhibits excellent specificity and sensitivity and is capable of discriminating infection of Plasmodium vivax, distinguishing the kinds of Plasmodia, and identifying mixed malaria infection with ease and economically. Further, the inventors developed a diagnosis kit including monoclonal and polyclonal antibodies to lactate dehydrogenase of P. vivax and monoclonal antibodies to lactate dehydrogenase of Plasmodium falciparum.
  • the present invention provides a method of detecting dual infection of malaria and a kit for diagnosing the same by preparing a specific monoclonal antibody to P. vivax lactate dehydrogenase (LDH) and simultaneously using a specific monoclonal antibody to P. falciparum LDH.
  • LDH lactate dehydrogenase
  • An aspect of the present invention provides a kit for diagnosing malaria, including specific monoclonal and polyclonal antibodies to P. vivax lactate dehydrogenase, a specific monoclonal antibody to P. falciparum lactate dehydrogenase, and a simultaneously specific monoclonal antibody to P. vivax lactate dehydrogenase and P. falciparum lactate dehydrogenase.
  • Another aspect of the present invention provides a method of diagnosing malaria using the kit.
  • a further aspect of the present invention provides hybridoma producing the monoclonal antibodies.
  • Yet another aspect of the present invention provides novel monoclonal antibodies specific to the lactate dehydrogenases used in the kit.
  • the present invention provides a kit for diagnosing malaria including specific monoclonal and polyclonal antibodies to lactate dehydrogenase of P. vivax, a specific monoclonal antibody to lactate dehydrogenase of P. falciparum, and a simultaneously specific monoclonal antibody to P. vivax and P. falciparum lactate dehydrogenases.
  • the diagnosis kit of the invention is a rapid and convenient kit which has excellent specificity and sensitivity over conventional diagnosis kits and is capable of distinguishing the kinds of malaria, in particular determining dual infection of malaria.
  • the monoclonal antibodies used in the kit for diagnosing malaria of the invention are ones specific to lactate dehydrogenase of P. vivax or to lactate dehydrogenase of P. falciparum, or both lactate dehydrogenase of P. vivax and lactate dehydrogenase of P. falciparum.
  • the monoclonal antibodies used in the kit of the invention include a monoclonal antibody 1H3C10 specific to lactate dehydrogenase of P. vivax produced by hybridoma of Accession No. KCTC11239BP, a monoclonal antibody T12E specific to lactate dehydrogenase of P. falciparum produced by hybridoma of Accession No.
  • KCTC11240BP and a monoclonal antibody T28C simultaneously specific to lactate dehydrogenase of P. vivax and the lactate dehydrogenase of P. falciparum produced by hybridoma of Accession No. KCTC11241BP.
  • the inventors of the present invention extracted DNA from malaria Plasmodia in the blood of a Korean patient infected with Plasmodium vivax and in the blood a Vietnamese patient infected with Plasmodium falciparum, respectively, and amplified the lactate dehydrogenase genes of P. vivax and P. falciparum by polymerase chain reaction (PCR), respectively, which were used to prepare two kinds of genetically recombinant antigens (Figs. 4 and 5).
  • PCR polymerase chain reaction
  • the genetically recombinant antigens were expressed in E coli, followed by refining (Fig. 6), after which they were injected into mice as immunogens to separate hybridoma secreting monoclonal antibodies specifically reactive only to lactate dehydrogenase of P. vivax, hybridoma secreting monoclonal antibodies specifically reactive only to lactate dehydrogenase of P. falciparum, and hybridoma secreting monoclonal antibodies specifically reactive to both lactate dehydrogenases of P. falciparum and P. vivax, thereby preparing monoclonal antibodies.
  • the prepared monoclonal antibodies antibodies exhibiting the highest reactivity, i.e., the specific antibody 1H3C10 to P.
  • kits may further include instructions, usually provided with a general diagnosis kit, diagnostic reagents, etc. in addition to the antibodies.
  • kits of the present invention includes Enzyme-Linked Immunosorbent Assay (ELISA), dipstick immunoassay, immunochromatographic assay, separate radioimmunoassay, flow-through immunoassay, etc.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • the kit of the present invention is a diagnosis kit in a strip form or a device form using immunochromatographic assay.
  • Immunochromatographic assay also referred to as a rapid test due to its convenient and rapid features, is based on a principle in which a malaria antigen in the serum of a blood sample reacts with a tracer antibody bound to a colloidal gold particle and then combines with a capture antibody settled on the inner surface of pores of a nitrocellulose membrane to form a colorized band while transferring through the pores by a capillary phenomenon, thereby identifying positivity or negativity with the naked eye. Because of its simple procedures and rapid results, the immunochromatographic assay is widely used in detecting hormones, antigens, antibodies, and pharmaceutical components.
  • an antigen-antibody complex is detected by a color particle coupling method, in which examples of color particles include a colloidal gold particle, colored glass, or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • color particles include a colloidal gold particle, colored glass, or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • the colloidal gold particle is used.
  • the capture antibody binding to the antigen-antibody complex employs a monoclonal antibody specific to lactate dehydrogenase of P. vlvax and a monoclonal antibody specific to lactate dehydrogenase of P. falciparum, preferably the monoclonal antibody 1H3C10 produced by the hybridoma of Accession No. KCTC11239BP and the monoclonal antibody T12E produced by the hybridoma of Accession No. KCTC11240BP.
  • Another example of the capture antibody is rabbit polyclonal antibodies specific to lactate dehydrogenase of P. vivax and the lactate dehydrogenase of P. falciparum, respectively.
  • a monoclonal antibody simultaneously specific to both P. vivax and P. falciparum lactate dehydrogenases, specifically, T28C produced by the hybridoma of Accession No. KCTC11241BP is used.
  • the kit for diagnosing malaria using the immunochromatographic assay comprises two main parts, that is, a nitrocellulose membrane with three invisible lines formed on the surface thereof and a glass fiber pad (or a plastic well) configured to hold an antibody-gold conjugate in a dry state.
  • Three different antibodies are fixed in the invisible lines of the nitrocellulose membrane.
  • the lowermost test line 1(Tl) holds the monoclonal antibody T12E specific to lactate dehydrogenase of P. falciparum
  • test line 2 (T2) holds the monoclonal antibody 1H3C10 specific to lactate dehydrogenase of P. vivax
  • the uppermost control line (C) holds a goat anti-mouse immunoglobulin (IgG) antibody.
  • the antibody-gold conjugate obtained by conjugation of the monoclonal antibody T28C simultaneously specific to both P. vivax and P. falciparum lactate dehydrogenases and colloidal gold particles is injected into a glass fiber pad (or a plastic well) and dried. If a sample is put in a sample depositary part of the kit thus constituted, the antibody-gold conjugate maintained in a dry state is hydrated and combines with an antigen in the sample while transferring through pores in the nitrocellulose membrane by a capillary phenomenon. Subsequently, the antigen binding to the antibody-gold conjugate reacts with the antibodies held in the invisible lines so that a corresponding invisible line appears red due to the antibody-gold conjugate with red.
  • the goat anti- mouse IgG antibody held in the upper line is capable of reacting with the antibody-gold conjugate even without an antigen, the control line appears red in every test, and thus, it can be verified that the test is carried out properly. That is, if an antigen is present, the antigen will react with a corresponding antibody in a test line of the kit, so that the test line and the control line appear at the same time. If there is no antigen, the test line will not appear but only the control line will turn into red.
  • the diagnosis kit of the invention simultaneously employs the monoclonal antibody specific to lactate dehydrogenase of P. vivax and the monoclonal antibody specific to lactate dehydrogenase of P. falciparum, thereby distinguishing the kinds of malarial Plasmodia and the mixed malaria infection. If the diagnosis kit with the aforementioned element is brought into contact with a P. falciparum positive sample, a color band appears on the test line 1 (Tl); and if the kit is brought into contact with a P. vivax positive sample, the color band appears on the test line 2 (T2). If the kit is brought into contact with a sample infected with both, the color band appears on the test lines 1 and 2 (Tl and T2) at the same time. Hence, the kit of the invention can identify the mixed malaria infection and discriminate the kinds of Plasmodia with convenience and rapidity.
  • Yet another aspect of the present invention provides a method for diagnosing the mixed malaria infection and discriminating the kinds of Plasmodia using rabbit polyclonal antibodies specific to P. vivax lactate dehydrogenase and P. falciparum lactate dehydrogenase, which are prepared by immuno-aff inity chromatography which employs the recombinant P. vivax lactate dehydrogenase and the recombinant P. falciparum lactate dehydrogenase according to the present invention. That is, as capture antibodies, a specific rabbit polyclonal antibody to P. falciparum lactate dehydrogenase, a specific rabbit polyclonal antibody to P.
  • vivax lactate dehydrogenase and a goat anti-mouse InG antibody are fixed to test line 1 (Tl), test line 2 (T2), and control line (C), respectively.
  • Colloidal gold particles are conjugated with a mouse monoclonal antibody T28C simultaneously specific to P. vivax and P. falciparum lactate dehydrogenases and disposed in a conjugation pad, thereby discriminatively diagnosing malaria.
  • Yet another aspect of the present invention provides a method of diagnosing malaria comprising performing an antigen-antibody reaction using the kit of the present invention.
  • collected whole blood is brought into contact with a sample depositary part of the kit and absorbed to determine the presence of P. falciparum or P. vivax in accordance with indications of test lines of the kit.
  • Yet another aspect of the present invention provides novel monoclonal antibodies and polyclonal antibodies specific to P. vivax and P. falciparum lactate dehydrogenases used in the kit of the invent ion .
  • Yet another aspect of the present invention provides a monoclonal antibody specific to P. vivax lactate dehydrogenase and a kit for diagnosing P. vivax comprising the same.
  • Yet another aspect of the present invention provides a method of detecting the mixed malaria infection and a kit for diagnosing the same using a specific monoclonal antibody to lactate dehydrogenase of P. vivax and a specific monoclonal antibody to lactate dehydrogenase of P. vivax, the kit further employing a monoclonal antibody simultaneously specific to P. vivax and P. falciparum lactate dehydrogenases.
  • the monoclonal antibodies and polyclonal antibodies can be prepared by any known method in the art.
  • the monoclonal antibodies are prepared as follows: a mouse is immunized with P. vivax and P. falciparum lactate dehydrogenases as immunogens; its splenocyte is fused with myeloma to reproduce hybridoma; and hybridoma are selected based on the specificity to the respective lactate dehydrogenases.
  • DNA is extracted from malarial Plasmodium in patient's blood infected with P. falciparum and from one infected with P. vivax, respectively, as illustrated in Fig.
  • Hybridoma are selected which secrete a monoclonal antibody reactive in the plate binding to the recombinant P. vivax lactate dehydrogenase but not reactive in the plate binding to the recombinant P. falciparum lactate dehydrogenase.
  • the monoclonal antibody thus secreted was named 1H3C10.
  • Hybridoma are selected which secrete a monoclonal antibody reactive in the plate binding to the recombinant P. falciparum lactate dehydrogenase but not reactive in the plate binding to the recombinant P. vivax lactate dehydrogenase.
  • the monoclonal antibody thus secreted was named T12E.
  • Hybridoma are selected which secrete a monoclonal antibody reactive in both plates.
  • the monoclonal antibody thus secreted was named T28C.
  • These monoclonal antibodies are employed in preparing the kit of the present invention.
  • the hybridoma thus selected are abdominally injected into mice, respectively, and, after a predetermined period of time, the ascitic fluid is extracted, followed by separation of monoclonal antibodies.
  • Hybridoma which produce the monoclonal antibody 1H3C10 selectively identifying only lactate dehydrogenase of P. vivax were accepted as KCTC11239BP (Acceptance Date: Nov.15, 2007), hybridoma which produce the monoclonal antibody T12E selectively identifying only lactate dehydrogenase of P.
  • Fig. 1 is a flowchart illustrating a method of producing a mouse monoclonal antibody against lactate dehydrogenase of Plasmodium vivax according to the present invention
  • Fig. 2 is a flowchart illustrating a method of producing a rabbit polyclonal antibody against lactate dehydrogenase of P. vivax according to the present invention
  • Fig. 3 is a DNA sequence of a primer used for gene amplification of P. vivax and P. falciparum lactate dehydrogenases according to the present invention
  • Figs. 4 and 5 are a DNA sequence of lactate dehydrogenase of P. vivax and a DNA sequence of lactate dehydrogenase of P. falciparum which are used to produce recombinant proteins according to the present invention, respectively!
  • Fig. 6 illustrates results of genetic amplification of lactate dehydrogenase of P. vivax of and lactate dehydrogenase of P. falciparum according to the present invention!
  • Fig. 7 illustrates the results obtained by purifying a recombinant protein, a gene of which has been expressed in E. coli.
  • M is standard size markers for the analysis of protein on the basis of molecular weight
  • lane 1 is a cell effluent where P. vivax lactate dehydrogenase is expressed
  • lane 2 is purified P. vivax lactate dehydrogenase
  • lane 3 is a cell effluent where P. falciparum lactate dehydrogenase is expressed
  • lane 4 is purified P. falciparum lactate dehydrogenase!
  • Figs. 8 and 9 are graphs depicting reactivities of monoclonal antibodies on P. vivax lactate dehydrogenase and P. falciparum lactate dehydrogenase according to the present invention.
  • the reactivities were measured by treating the monoclonal antibodies of the invention having each different concentration with plates containing P. falciparum lactate dehydrogenase (Fig. 8) and P. vivax lactate dehydrogenase (Fig. 9), respectively!
  • Fig. 10 is graphs depicting dissociation constants of the monoclonal antibodies on P, vivax lactate dehydrogenase and P. falciparum lactate dehydrogenase according to the present invention!
  • Fig. 11 is a diagram illustrating a strip for an immunochromatographic assay that can rapidly diagnose infection of P. falciparum, P. vivax, or the mixed infection thereof according to the present invention!
  • Fig. 12 is pictures showing test results obtained by the strip in the rapid immunochromatographic assay diagnosing a malarial specific antigen according to the present invention!
  • Fig. 13 is a diagram illustrating a device for an immunochromatographic assay that can rapidly diagnose infection of P. falciparum, P. vivax, or mixed infection thereof according to the present invention! and Fig. 14 is pictures showing test results obtained by the device in the rapid immunochromatographic assay diagnosing a malarial specific antigen according to the present invention.
  • a portion corresponding to a Plasmodium lactate dehydrogenase gene was amplified using opposite end-primers, and then cloned to an expression vector pET-28a (Novagen Ltd., USA), capable of expression in E. CoIi.
  • the primer has a sequence given in Fig. 3. That is, in the amplification and cloning, primers BL-PvLDH-F (Sequence Number 1) and BL-PvLDH-R (Sequence Number 2) were used for P.
  • vivax lactate dehydrogenase and primers BL-PfLDH-F (Sequence Number 3) and BL- PfLDH-R (Sequence Number 4) were used for P. falciparum lactate dehydrogenase.
  • the cloned Plasmodium lactate dehydrogenase genes were analyzed by a DNA sequence analyzer, ABI 377 (PerkinElmer Ltd., USA), results of which are shown in Fig. 4 ⁇ P. vivax lactate dehydrogenase: Sequence Number 5) and Fig. 5 (P. falciparum lactate dehydrogenase: Sequence Number 6). 2) Purification of recombinant P. vivax and P.
  • Plasmodium lactate dehydrogenases were purified using histidine-aff inity columns (Invitrogen Ltd., USA) and treated with thrombin (Novagen Ltd., USA) to separate a histidine tag. The product was passed through the histidine-affinity column again to eliminate the histidine tag, and passed through a HiTrap Benzamidine column (Amersham Bioscience, Sweden) to eliminate the thrombin, thereby producing pure recombinant Plasmodium lactate dehydrogenases.
  • a suspension was prepared by mixing a phosphate buffered saline (PBS) solution containing a certain amount of the recombinant Plasmodium lactate dehydrogenases with the same volume of a complete Freund's adjuvant and subcutaneousIy injected into a goat, a rabbit, or other animals prone to produce a polyclonal antibody. After three weeks, the subcutaneous injection was carried out again in the same manner with an incomplete Freund's adjuvant instead, and after another three weeks the injection was repeated in the same manner. Three to five days after the last immunization, blood was collected to obtain a serum, which was diluted with a PBS solution (1/1000) to determine the titer of an antibody by enzyme-linked immunosorbent assay (ELISA). If the titer was low, immunization was carried out one week later.
  • PBS phosphate buffered saline
  • Blood was collected from thoroughly immunized subject and centrifuged to obtain a serum.
  • the serum was injected into a column containing a Sepharose filler binding to the recombinant lactate dehydrogenase via a covalent bond.
  • Five times or more of physiological saline solution as much volume as fillers in the column was used to wash away proteins that were not bound to the column.
  • antibodies combined to the column were eluted with a 10OmM glycine solution (pH: 2.8).
  • a IM Tris solution pH: 9.0
  • the eluted antibody solution was concentrated and dialyzed in a 15OmM phosphate buffer. Then, the amount of the antibodies was determined using a Bradford assay and the antibodies were stored in a freezer until use.
  • a suspension was prepared by mixing a PBS solution containing 100/zg of each of the recombinant Plasmodium lactate dehydrogenases with the same volume of a complete Freund's adjuvant and injected into a six to eight-week-old female mouse's peritoneal (BALA/C, Dae Han Biol ink Co., Ltd, Korea). After two weeks, the peritoneal injection was carried out again in the same manner with an incomplete Freund's adjuvant instead, and after another two weeks the injection was repeated in the same manner. Two days later after the last immunization, blood was collected from the tail to obtain a serum, which was diluted with a PBS solution (1/1000) to determine the titer of an antibody by ELISA. If the titer was low, immunization was carried out two weeks later.
  • RPMI1640 Five minutes later, ImI of RPMI1640 was added over 30 seconds, and then another 3ml was added over 30 seconds. Subsequently, 17ml of RPMI1640 was added over one minute, and another 20ml more to react for five minutes. After centrifugal separation at 200Xg for five minutes, the medium was removed. The product was suspended carefully in 50ml of RPMI1640 containing 1% Hypoxanthine Aminopterin Thymidine medium (HAT), after which fused cells were aliquoted at 100/ ⁇ /well in a 96-well cell culture plate containing feeder cells and cultured at 37 ° Cin a 5% CO2 incubator.
  • HAT Hypoxanthine Aminopterin Thymidine medium
  • 0.5ml of an incomplete Freund's adjuvant was injected into the abdominal cavity of a six to eight-week-old mouse (BALA/C). Suspending hybr idoma in a PBS solution and cell counting on day seven, 1.5X10 6 cells per mouse were suspended in 0.5ml of a PBS solution and injected into a mouse's peritoneal. When being produced sufficiently after one or two weeks, the ascitic fluid was collected with an injector and kept in a freezer.
  • Ammonium sulfate was added to the collected ascitic fluid at a concentration of 10% and mixed for 30 minutes. Then, the product was centrifuged at 15,000 rpm for 30 minutes to collect a supernatant. Ammonium sulfate was added to the supernatant at a concentration of 50% and left at 4 ° C for 30 minutes. Centrifuging the product at 15,000 rpm for 30 minutes, a supernatant was discarded and a precipitant was suspended in a 2OmM phosphate buffer (pH: 7.0). The suspension was dialyzed in a 2OmM phosphate buffer for over 18 hours.
  • the dialyzed solution was injected to a protein G-coupled column and equilibrated by a 2OmM phosphate buffer (pH: 7.0), after which unabsorbed materials were removed using a phosphate buffer.
  • Antibodies attached to the column were eluted with a 10OmM glycine solution (pH: 2.8).
  • a IM Tris solution (pH: 9.0) was added at a volume of 1/10 of the eluted solution to adjust pH.
  • the eluted antibody solution was concentrated and dialyzed in a 15OmM phosphate buffer. Then, the amount of the antibodies was determined using a Bradford assay and the antibodies were stored in a freezer before use.
  • the refined Plasmodium lactate dehydrogenase-specif ic monoclonal antibodies were measured by ELISA to identify reactivity with respect to Plasmodium lactate dehydrogenases.
  • 96-well ELISA plates were coated with two kinds of Plasmodia lactate dehydrogenases at a concentration of 5 mg/ml , respectively.
  • the refined Plasmodium lactate dehydrogenase-specif ic monoclonal antibodies were diluted in each step to react with a plate coated with P. vivax lactate dehydrogenase and a plate coated with P. falciparum lactate dehydrogenase, respectively, and uncombined antibodies were removed by washing.
  • a goat ant i-mouse immunoglobulin (IgG) antibody was reacted with horseradish peroxidase (HRP), and unreacted peroxidase-IgG was removed by washing.
  • Tetramethyl benzidine (TMB) serving as a substrate of peroxidase, was added to give color, and absorbance (A450) was measured according to reactivity.
  • 1H3C10 exhibited good reactivity with respect to P. vivax lactate dehydrogenase
  • T12E exhibited good reactivity with respect to P. falciparum lactate dehydrogenase
  • T28C exhibited good reactivity with respect to both.
  • the Klotz plot was used via indirect competitive ELISA to obtain the results given in Fig. 10. That is, the monoclonal antibody T12E has a dissociation constant of 25.24+ 0.51X10 '9 ; the monoclonal antibody T28C has a dissociation constant of 24.21 ⁇ 2.07xl0 ⁇ 9 ; and the monoclonal antibody 1H3C10 has a dissociation constant of
  • the specific monoclonal antibody T12E to P. falciparum lactate dehydrogenase, the specific monoclonal antibody 1H3C10 to P. vivax lactate dehydrogenase, and the goat anti-mouse immunoglobulin (IgG) antibody were placed on specific positions of a nitrocellulose membrane attached to a plastic card, i.e., on test line 1 (Tl), test line 2 (T2), and control line (C), respectively, and dried in an incubator at 30 ° C for two days, so that they could be completely settled on the nitrocellulose membrane.
  • Tl test line 1
  • T2 test line 2
  • C control line
  • the monoclonal antibody T28C simultaneously specific to both
  • P. vivax and P. falciparum lactate dehydrogenases was mixed with colloidal gold particles of about 40 nm and reacted in a 371water bath for one hour so that the monoclonal antibody was bound to the colloidal gold particles. Then, 3% and 1% bovine serum albumin (BSA) and sucrose were added thereto, respectively, and reacted for one hour with colloidal gold particles which had not bound to the antibody. Subsequently, the product was centrifuged at 10,000 rpm to collect gold conjugates binding to the monoclonal antibody, which were measured for absorbance at 540 nm and kept refrigerated. The gold conjugates were injected into a plastic micro-well plate and dried, thereby preparing a gold conjugate well.
  • BSA bovine serum albumin
  • an absorbance pad capable of absorbing a sample and a buffer was attached to an upper portion of the plastic card to which the nitrocellulose membrane binding to a monoclonal antibody to Plasmodium lactate dehydrogenase was adhered, and a sticker was attached to the top thereof, thereby completing a strip kit .
  • Example 5 Preparation of Device Diagnosis Kit for Rapid Immunochromatographic Assay Using Monoclonal Antibodies against P. vivax and P. falciparum lactate dehydrogenases
  • an absorbance pad capable of absorbing a sample and a buffer was attached to an upper portion of the plastic card to which the nitrocellulose membrane binding to a monoclonal antibody to Plasmodium lactate dehydrogenase was adhered, and a sample pad having a sample deposited thereto and a conjugate pad were attached to lower portions thereof.
  • the assembled reaction strip was placed in a plastic housing, thereby completing a device kit, as shown in Fig. 13.
  • a gold conjugate well and a washing well were prepared.
  • One drop of a sample mobile phase was dropped in the gold conjugate well, and four drops thereof were dropped in the washing well provided with the kit.
  • 10ml of blood was collected with a capillary tube.
  • the collected blood was placed in the gold conjugate well and mixed with the sample mobile phase using a capillary tube.
  • the reaction strip was moved to the washing well containing a washing solution and kept for ten minutes. Once the strip have completely reacted, infection with P. falciparum or P.
  • vivax or the mixed malaria infection was determined according to a position of a red band appearing on the test lines, as illustrated in Fig. 12. Namely, it was determined that the sample was positive for P. falciparum when the color band appeared only on the test line 1 (Tl); the sample was positive for P. vivax when the color band appeared only on the line 2 (T2); and the sample was infected with mixed malaria when the color bands appeared on both lines.
  • the device was placed on a flat surface.
  • lO ⁇ A of blood was collected with a capillary tube.
  • the collected whole blood was placed in a sample depositary part to be absorbed, as shown in Fig. 13.
  • Three drops of a washing solution were dropped in a washing solution inlet and stored for 15 minutes.
  • the infection of P. falciparum or P. vivax or the mixed malaria infection was determined according to a position of a red band appearing on the test lines, as shown in Fig. 14. Namely, it was determined that the sample was positive for P. falciparum when the color band appeared only on the test line 1 (Tl); the sample was positive for P. vivax when the color band appeared only on the line 2 (T2); and the sample was infected with mixed malaria when the color bands appeared on both lines.
  • the diagnosis kits prepared according to Examples 3 to 5 were used to test 129 samples positive for P. vivax, 86 samples positive for P. falciparum, and 7 samples positive for mixed malaria.
  • a total of 129 P. r/raz-positive samples were tested, in which 30 samples were donated by Sungkyunkwan University of Medicine (Suwon, Korea), 47 samples by Ho Chi Minh City University of Medicine (Ho Chi Minh City, Vietnam), and 52 samples by Inje University of Medicine (Busan, Korea).
  • falciparum positive samples were tested, in which 25 samples were donated by Ho Chi Minh City University of Medicine (Ho Chi Minh City, Vietnam), 9 samples by Inje University of Medicine, and 52 samples by National Institute of Malariology, Parasitology and Entomology (Hanoi, Vietnam).
  • the test results are given in Table 1 (Comparative test results of samples positive for P. vivax), Table 2 (Comparative test results of samples positive for P. falciparum) , and Table 3 (Comparative test results of samples positive for mixed infection of P. vivax and P. falciparum) .
  • the diagnosis kit of the invention had a superior sensitivity to conventional diagnosis kits in view of a sensitivity of 85 to 95% with respect to P. falciparum and a sensitivity of 90% or less with respect to P. vivax. Further, the kit of the present invention could detect mixed malaria infection, which could not have been detected by the conventional kits, and thus excellent performance of the kit according to the invention was confirmed.
  • the diagnosis kit having antibodies for P. vivax and P. falciparum lactate dehydrogenases realized by the present invention is a convenient and inexpensive kit, which can discriminate P. vivax and P. falciparum, diagnose mixed infection of P. vivax and P. falciparum, which cannot be diagnosed by the conventional kits, and overcome a low sensitivity to P. vivax.
  • the present invention will have a global impact on control of bacteria resistant to P. vivax medication.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur des trousse de diagnostic de la malaria comprenant un anticorps monoclonal spécifique de la LDH du Plasmodium vivax, un anticorps monoclonal spécifique de la LDH du Plasmodium falciparum, un anticorps monoclonal spécifique des LDHs du Plasmodium vivax et du Plasmodium falciparum, et sur une méthode de diagnostic d'une infection mixte de la malaria les utilisant. Cette trousse est la première trousse rapide et pratique du monde à pouvoir différencier différentes sortes de malarias et diagnostiquer une infection mixte par le P. vivax et le P. falciparum avec une grande spécificité et une grande sensibilité. L'invention porte en outre: sur les anticorps monoclonaux se fixant spécifiquement aux LDHs du P. vivax et du P. falciparum, utilisés dans la trousse; sur les hybridomes les produisant; et sur une trousse de diagnostic de la malaria comprenant un anticorps monoclonal spécifique du P. vivax.
PCT/KR2007/006962 2007-12-13 2007-12-28 Trousse de diagnostic d'une infection mixte de malaria par le plasmodium vivax et le plasmodium falciparum utilisant des anticorps spécifiques de la lactate déshydrogénase du plasmodium vivax et du plasmodium falciparum WO2009075404A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020070130495A KR100968848B1 (ko) 2007-12-13 2007-12-13 삼일열 및 열대열 말라리아 젖산탈수소효소에 특이적인항체 및 이를 포함하는 열대열 말라리아와 삼일열말라리아의 중복 감염 진단 키트
KR10-2007-0130495 2007-12-13

Publications (1)

Publication Number Publication Date
WO2009075404A1 true WO2009075404A1 (fr) 2009-06-18

Family

ID=40755627

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2007/006962 WO2009075404A1 (fr) 2007-12-13 2007-12-28 Trousse de diagnostic d'une infection mixte de malaria par le plasmodium vivax et le plasmodium falciparum utilisant des anticorps spécifiques de la lactate déshydrogénase du plasmodium vivax et du plasmodium falciparum

Country Status (2)

Country Link
KR (1) KR100968848B1 (fr)
WO (1) WO2009075404A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102229913A (zh) * 2011-05-26 2011-11-02 南方医科大学 一种用于检测疟原虫的胶体金快速免疫层析检测试纸及抗体和细胞株
EP2532749A1 (fr) * 2011-06-07 2012-12-12 Postech Academy-industry Foundation Liaison spécifiquement d'aptamère d'ADN (déshydrogénase de lactacte de Plasmodium)
WO2017027677A1 (fr) * 2015-08-11 2017-02-16 University Of Central Florida Research Foundation, Inc. Dispositif capteur de surveillance passive d'insectes
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
WO2020049444A1 (fr) * 2018-09-03 2020-03-12 Module Innovations Private Limited Dispositif et procédé de dosage à écoulement latéral pour identification différentielle d'espèces de plasmodium

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102002104B1 (ko) * 2018-05-16 2019-07-19 원광대학교산학협력단 열대열 말라리아원충의 젖산탈수소효소에 특이적으로 결합하는 펩티드 및 이의 용도
CN111378034B (zh) * 2018-12-29 2021-10-12 东莞市朋志生物科技有限公司 抗恶性疟原虫hrp-ii抗体
KR20220111445A (ko) 2021-02-02 2022-08-09 최해용 방역 워크 부스
CN113897339B (zh) * 2021-09-23 2024-01-09 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) 小鼠杂交瘤细胞及用于区分检测恶性疟、间日疟、三日疟、卵形疟的快速诊断试条

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016354A2 (fr) * 2001-08-16 2003-02-27 Institut Pasteur Anticorps de recombinaison anti-plasmodium falciparum
US20040132117A1 (en) * 2000-02-17 2004-07-08 Kook-Jin Lim Immunoassay and diagnostic reagent for malaria
KR100559370B1 (ko) * 2005-05-27 2006-03-15 아산제약 주식회사 말라리아 젖산탈수소효소 및 알돌라아제에 특이적인 단클론항체를 포함하는 말라리아 진단 키트

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040132117A1 (en) * 2000-02-17 2004-07-08 Kook-Jin Lim Immunoassay and diagnostic reagent for malaria
WO2003016354A2 (fr) * 2001-08-16 2003-02-27 Institut Pasteur Anticorps de recombinaison anti-plasmodium falciparum
KR100559370B1 (ko) * 2005-05-27 2006-03-15 아산제약 주식회사 말라리아 젖산탈수소효소 및 알돌라아제에 특이적인 단클론항체를 포함하는 말라리아 진단 키트

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102229913A (zh) * 2011-05-26 2011-11-02 南方医科大学 一种用于检测疟原虫的胶体金快速免疫层析检测试纸及抗体和细胞株
EP2532749A1 (fr) * 2011-06-07 2012-12-12 Postech Academy-industry Foundation Liaison spécifiquement d'aptamère d'ADN (déshydrogénase de lactacte de Plasmodium)
CN102816763A (zh) * 2011-06-07 2012-12-12 浦项工科大学校产学协力团 特异性结合于疟原虫乳酸脱氢酶pLDH的DNA适体
JP2012254074A (ja) * 2011-06-07 2012-12-27 Postech Academy-Industry Foundation ｐＬＤＨに特異的に結合するＤＮＡアプタマー
US8541561B2 (en) 2011-06-07 2013-09-24 Postech Academy-Industry Foundation DNA aptamer specifically binding to pLDH (plasmodium lactate dehydrogenase)
CN102816763B (zh) * 2011-06-07 2014-07-16 浦项工科大学校产学协力团 特异性结合于疟原虫乳酸脱氢酶pLDH的DNA适体
WO2017027677A1 (fr) * 2015-08-11 2017-02-16 University Of Central Florida Research Foundation, Inc. Dispositif capteur de surveillance passive d'insectes
US10948491B2 (en) 2015-08-11 2021-03-16 University Of Central Florida Research Foundation, Inc. Passive insect surveillance sensor device
US11693004B2 (en) 2015-08-11 2023-07-04 University Of Central Florida Research Foundation, Inc. Passive insect surveillance sensor device
WO2020049444A1 (fr) * 2018-09-03 2020-03-12 Module Innovations Private Limited Dispositif et procédé de dosage à écoulement latéral pour identification différentielle d'espèces de plasmodium
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
CN110146697B (zh) * 2019-06-03 2022-02-11 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

Also Published As

Publication number Publication date
KR100968848B1 (ko) 2010-07-09
KR20090062991A (ko) 2009-06-17

Similar Documents

Publication Publication Date Title
WO2009075404A1 (fr) Trousse de diagnostic d'une infection mixte de malaria par le plasmodium vivax et le plasmodium falciparum utilisant des anticorps spécifiques de la lactate déshydrogénase du plasmodium vivax et du plasmodium falciparum
Sarkari et al. Immunodiagnosis of human fascioliasis: an update of concepts and performances of the serological assays
AU2010200492B2 (en) Method and device for trichomonas detection
Lagatie et al. Evaluation of the diagnostic performance of Onchocerca volvulus linear epitopes in a peptide enzyme-linked immunosorbent assay
Maddison Serodiagnosis of parasitic diseases
Fargeas et al. Synthetic peptide-based enzyme-linked immunosorbent assay for serodiagnosis of visceral leishmaniasis
Ashrafmansouri et al. Utility of western blot analysis for the diagnosis of cutaneous leishmaniasis
KR100982064B1 (ko) 말라리아 원충에 대한 단일클론 항체 및 이를 이용한말라리아의 진단방법
US8173383B2 (en) Methods and materials for the detection of Leishmania infection
Pinedo-Cancino et al. The efficacy of L.(L.) chagasi excreted-secreted antigens (ESAs) for visceral leishmaniasis diagnosis is due to low levels of cross-reactivity
KR100559370B1 (ko) 말라리아 젖산탈수소효소 및 알돌라아제에 특이적인 단클론항체를 포함하는 말라리아 진단 키트
WO2009069942A2 (fr) Trousses de diagnostic rapide pour la détection d'anticorps dirigés contre paragonimus westermani et anticorps monoclonaux destinés à la production d'antigènes spécifiques de paragonimus westermani
Sarkari et al. A comparative study of antigen and antibody detection in visceral leishmaniasis using serum and urine-based ELISA
Park et al. Development and evaluation of an immunochromatographic kit for the detection of antibody to Plasmodium vivax infection in South Korea
Hommel et al. The direct agglutination test: a non-specific test specific for the diagnosis of visceral leishmaniasis?
EP3635401A1 (fr) Dosage sérologique pour le dépistage d'une infection paludéenne récente
KR102002104B1 (ko) 열대열 말라리아원충의 젖산탈수소효소에 특이적으로 결합하는 펩티드 및 이의 용도
Ayi et al. Development of membrane-based tests for the detection of urinary antigens and antibodies in human toxoplasmosis: preliminary studies in Ghanaian patients
Khusmith et al. Two-site sandwich ELISA for detection of Plasmodium vivax blood stage antigens using monoclonal and polyclonal antibodies
WO2009130709A2 (fr) Antigène membranaire non recombinant, kit de diagnostic l'employant et procédé de détection de la leishmaniose viscérale et de la pkdl
Chakraborti et al. Immune complex antigens as a tool in serodiagnosis of kala-azar
Feng Immunodiagnosis of human and canine echinococcosis and community studies in northwestern China
EP3667322A1 (fr) Détection sérologique d'anticorps de plasmodium
Rajesh et al. New antigenic approach to diagnose visceral leishmaniasis in India
CN116990507A (zh) 一种用于猴痘病毒感染的早期筛查的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07860742

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07860742

Country of ref document: EP

Kind code of ref document: A1