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WO1997024141A1 - Anticorps monoclonaux et methode de capture immunologique pour la quantification et la differenciation d'especes d'hematozoaires - Google Patents

Anticorps monoclonaux et methode de capture immunologique pour la quantification et la differenciation d'especes d'hematozoaires Download PDF

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WO1997024141A1
WO1997024141A1 PCT/US1996/020672 US9620672W WO9724141A1 WO 1997024141 A1 WO1997024141 A1 WO 1997024141A1 US 9620672 W US9620672 W US 9620672W WO 9724141 A1 WO9724141 A1 WO 9724141A1
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plasmodium
lactate dehydrogenase
monoclonal antibody
pldh
diagnostic method
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Robert C. Piper
Michael T. Makler
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • TITLE MONOCLONAL ANTIBODIES AND IMMUNO-CAPTURE METHOD FOR QUANTITATION AND SPECIATION OF MALARIA PARASITES BACKGROUND OF THE INVENTION
  • Malaria a protozoan disease transmitted by the Anopheles mosquito, affects millions of people each year and has been characterized as "the most important of all infectious diseases" (Sir Macfarlane Burnet).
  • Malaria a protozoan disease transmitted by the Anopheles mosquito, affects millions of people each year and has been characterized as "the most important of all infectious diseases" (Sir Macfarlane Burnet).
  • Malaria remains endemic in many countries including Africa, Central America, the Caribbean Islands, South America, Europe, Asia, Indian sub-continent, Eastern Asia and Oceania. The disease is characterized by fever, rigors, anemia, splenomegaly, and chronic relapsing course.
  • the disease can be caused by four species of protozoa: Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium falciparum.
  • Plasmodia go through a number of morphological changes with multiplication of the organisms in man and in insect vectors. Man is infected by the saliva of a female anopheline mosquito that contains the infective sporozoite form. This sporozoite then enter the liver cells where they multiply. After approximately seven to ten days, multiple small forms break out of the liver cells into the blood and subsequently enter red blood cells. The dormancy of the infection in the liver cells explain relapses in malaria caused by Plasmodium vivax and Plasmodium ovale .
  • Another diagnostic procedure involves the use of thin blood smears. While thin smears are easier to examine, the scarceness of cells in a thin smear increases the likelihood that a diagnosis will be missed. Additionally, because the intensity of parasitemia varies greatly from hour to hour, frequent repeated examinations of thin blood smears may be required which may be prohibitively labor intensive and expensive, especially in endemic areas.
  • antiplasmodial antibodies are produced in animals during a malarial infection.
  • MAbs monoclonal antibodies
  • Plasmodium voelii a rodent malarial parasite.
  • MAbs identified monoclonal antibodies (MAbs) to Plasmodium voelii , a rodent malarial parasite.
  • MAbs identified stage specific, species specific, and cross-reactive antigens. The antigens were found on the surface or within the cytoplasm of the parasite, but not on the surface of erythrocytes from infected animals.
  • LDH parasitic LDH
  • This method comprises an enzyme assay which measures the ability of Plasmodium LDH (pLDH) to rapidly use 3-acetyl pyridine NAD as a coenzyme in the reaction leading to the formation of pyruvate from lactate.
  • pLDH Plasmodium LDH
  • Kaushal, et. al. demonstrated that polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to LDH of P. knowlesi .
  • Kaushal, D.C. et al. "Antibodies to Lactate Dehydrogenase of Plasmodium Knowlesi are Specific to Plasmodium Species", Immunological Investigations , 17:507-516 (1988).
  • This invention discloses an improved method for the detection and quantitation of malarial infection.
  • a series of monoclonal antibodies has been developed that can capture an active metabolic enzyme, specifically Plasmodium lactate dehydrogenase (pLDH).
  • the monoclonal antibodies are specific for Plasmodium LDH and do not cross-react with LDH isoforms present in whole blood of human, bovine, murine, avian, or other primate sources of LDH.
  • the activity of the captured pLDH may then be assayed using standard assay procedures which are well known to those of ordinary skill in the art.
  • the pLDH is assayed using the LDH substrate lactate and 3-acetyl pyridine nicoti ⁇ amide adenine dinucleotide as specified in U.S. Patent 5,124,141 dated June 23, 1992, the disclosure of which is hereby incorporated by reference.
  • This novel assay method has increased the specificity of the pLDH assay as recorded in Patent No. 5,124,141 by ten to one hundred fold.
  • the present invention also encompasses the development of a panel of monoclonal antibodies capable of differentiating LDH isoforms from different species of Plasmodium that infect humans. These species include P . falciparum, P . vivax, P . ovale, and P . malariae .
  • a subset within this panel of monoclonal antibodies can capture pLDH from the human-specific Plasmodium species in the fully active form. Binding competition studies used to epitope map the antibodies show that the monoclonal antibodies bind to sites distinct from one another.
  • the present invention further involves a test based upon the specificity of the monoclonal antibodies which may be used to differentiate between different species of malaria-causing parasites in humans as well as Plasmodium from murine, avian, and primate sources. Since correct speciation is a critical feature for diagnostic and epidemiological studies of malaria, this new set of tests is extremely useful for the efficient and timely diagnosis of malaria. These tests are based upon the fact that the monoclonal antibodies directed to pLDH are able to capture these active parasitic enzymes from the sample, whether this be hemolyzed red cells, plasma, serum, saliva, or other body fluids.
  • the invention includes several enzyme-immuno assay formats which are capable of capturing pLDH from different Plasmodium species.
  • Figure 1 is a graph demonstrating the column chromatography of pLDH using Cibacron blue sepharose chromatography from culture lysates.
  • pLDH activity APAD
  • human red blood cell LDH (1st NAD peak) because pLDH binds tightly to Cibracron blue (Column profile).
  • Figure 2 is an agarose electrophoresis gel in which recombinant pLDH has been stained with NAD + , NBT and PES.
  • Figure 3 are microtiter plates which have been coated with the MAbs: 6C9a, 6C9b, 17E4, 19G7, and 7G9 which demonstrate that the plates are capable of capturing enzymatically active pLDH from blood samples.
  • Figure 4 is a graph showing the measurement of specific activities (mOD/min) of 7 samples of P. falciparum and 3 samples of P. vivax.
  • Figure 5 is a graph showing the quantitation of the ICpLDH assay demonstrating the parasitic activity (mOD/ml) plotted as a function of % parasitemia.
  • Figure 6 shows microtiter wells coated with either the 19G7 monoclonal antibody (pan-specific) or the 17E4 monoclonal antibody (P. falciparum specific).
  • the wells were washed 2 times with PBS and incubated with APAD containing NBT and Diaphorase. Either 100 ⁇ ls or 5 ⁇ ls of blood sample were incubated in a coated microtiter well for 30 minutes.
  • Figure 7 is a graph demonstrating the activity of pLDH as tested in the presence of the anti-pLDH antibodies 6C9a, 7G9, 17E4, and 19G7.
  • a solution of recombinant pLDH was incubated for five minutes with the indicated concentration of antibody ( ⁇ g/ml) prior to assay with APAD. Only the 6C9a antibody was inhibitory to the enzyme activity.
  • Figure 8 visually depicts the activity of the indicated Plasmodium species when incubated in microtiter wells coated with the indicated antibody.
  • the 6C9a antibody recognized all Plasmodium LDH isoforms.
  • Plasmodium lactate dehydrogenase the terminal enzyme of the glycotic pathway, has been demonstrated in several malarial parasites. This enzyme plays an important role in the regulation of intracellular redox state and thus affects the energy metabolism of these parasites. Although considerable work has been done to study the kinetic and electrophoretic behavior of Plasmodium LDH, little has been demonstrated about its antigenic nature.
  • the present invention is directly related to the antigenic nature of pLDH in the development of monoclonal antibodies and an immunocapture method for quantifying and speciating malarial parasites.
  • This invention is thus useful in the diagnosis and detection of malaria in humans caused by the human malarial parasites P. falciparum, P . vivax, P . ovale, and P . malariae .
  • the assay system is based upon the quantitation of previously captured pLDH.
  • This parasite enzyme assay can detect pLDH produced by all four species of Plasmodium that infect humans, thus making the specific and sensitive measure of pLDH a useful tool for the diagnosis of all forms of malaria. Because the measure of pLDH is quantitative and reflects the presence of viable parasites, such a measure is also useful to quickly test the efficacy of drug treatment for a particular malarial infection, to determine the presence of drug resistance, and to also detect the presence of parasites in blood products.
  • the invention sets forth a unique method which: (1) enhances the sensitivity and specificity of the measurement of pLDH; (2) relates the levels of pLDH whole blood to the percent parasitemia and the severity of the disease; (3) formats an easy to perform, simple to interpret, inexpensive, and quantitative diagnostic test for malaria using whole blood, lysed red blood cells, and plasma- serum; and (4) modifies the immuno-enzyme assay to reformat an in-vitro culture and sensitivity assay to evaluate multiple drug resistance under field conditions.
  • the invention thus embodies a method for detection of pLDH, a marker of active malaria causing Plasmodium parasites, and a method of diagnosis, therapy, or study of the same by assaying for the presence of pLDH.
  • the presence of the pLDH may be ascertained by collecting a biological sample which may include serum, plasma, urine, saliva, or other body fluids.
  • a preferred immunoassay uses a specialized detection reagent consisting of 3-acetyl pyridine adenine dinucleotide (APAD) in combination with a selected substrate, such as a lactate salt or lactic acid, and a buffer, as described in U.S. Pat. No. 5,124,141 incorporated herein by reference.
  • APAD 3-acetyl pyridine adenine dinucleotide
  • the combination of the reagent with a sample of parasite-infected blood catalyzes oxidation of the substrate and simultaneously reduces the APAD.
  • the host LDH has no influence on the APAD.
  • reduced APAD may be detected in numerous ways.
  • the reduced material may be spectrophotometrically detected by observing its characteristic absorption peak at about 363-365 nm, which is distinctive from the absorption spectra of a sample containing unreduced APAD.
  • the chromogens e.g. tetrazolium salts
  • the reduced APAD is fluorescent, and may be detected using an appropriately configured fluorescent lamp system or fluorometer.
  • Monoclonal antibodies are prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g., by fusion with myeloma cells or by Epstein-Barr virus transformation in screening for clones expressing the desired antibody.
  • hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.
  • the hybrid cell lines can be maintained in vitro in cell culture media.
  • the cell lines producing the antibodies can be selected and/or maintained in a medium containing hypoxanthine-aminopterin thymidine (HAT) .
  • HAT hypoxanthine-aminopterin thymidine
  • the hybrid cells lines can be stored and preserved in any number of conventional ways, including freezing and storage under liquid nitrogen. Frozen cell lines can be revived and cultured indefinitely with resumed synthesis and secretion of monoclonal antibody.
  • the secreted antibody is recovered from tissue culture supernatant or ascites fluid by conventional methods such as immune precipitation, ion exchange chromatography, affinity chromatography such as protein A/protein G column chromatography, or the like.
  • the antibodies described herein are also recovered from hybridoma cell cultures by conventional methods such as precipitation with 50% ammonium sulfate.
  • the purified antibodies can then be sterile filtered.
  • the term "monoclonal antibody” as used herein refers to any antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the monoclonal antibodies herein also include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an anti-pLDH antibody with a constant domain (e.g. "humanized” antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibodies fragment (e.g., Fab, F(ab')2 and Fv), so long as they exhibit the desired biological activity.
  • Fab, F(ab')2 and Fv antibodies fragment
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Koehler and Milstein, supra, or may be made by recombinant DNA methods (Cabilly, et al. supra).
  • Anti-pLDH antibodies are useful in diagnostic assays for pLDH expression in specific cells or tissues wherein the antibodies are labeled as described below and are immobilized on an insoluble matrix. Anti-pLDH antibodies also are useful for the affinity purification of the pLDH from recombinant cell culture or natural sources. The anti-pLDH antibodies that do not detectably cross react with other pLDH can be used to purify each pLDH free from other homologous receptors. Suitable diagnostic assays for the pLDH are well known per se.
  • a biological sample may be assayed for pLDH by obtaining the sample from a desired source, admixing the sample with anti-pLDH antibody to allow the antibody to form antibody/pLDH complex with any pLDH present in the mixture and detecting any antibody/pLDH complex present in the mixture.
  • the biological sample may be prepared for assay by methods known in the art which are suitable for the particular sample.
  • the methods of admixing the sample with antibodies and the methods of detecting antibodies/pLDH complex are chosen according to the type of assay used.
  • Such assays include competitive and sandwich assays, and steric inhibition assays.
  • Competitive and sandwich methods employ a phase-separation step as an integral part of the method while steric inhibition assays are conducted in a single reaction mixture.
  • Analytical methods for the pLDH all use one or more of the following reagents: labeled pLDH analog, immobilized pLDH analog, labels anti-pLDH antibody, immobilized anti-pLDH antibody and steric conjugate.
  • the label used is any detectable functionality that does not interfere with the binding of pLDH and anti-pLDH antibody.
  • Numerous labels are known for use in immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties such as enzymes, that must be reacted or derivatized to be detected.
  • Examples of such labels include the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyn, umbelliferone, luceriferases, e.g.
  • firefly lucifera and bacterial lucifera (U.S. Patent No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g.
  • glucose oxidase glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase
  • an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • coupling agents such as dialdehydes, carbondiimides, dimaleimides, bisimidates, bisdiazotized benzidine, and the like may be used to tag antibodies with the above described fluorescent, chemiluminescent and enzyme labels. See for e.g. U.S. Patent Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al.. Nature , 144:945 (1962); David et al. , Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol .
  • Preferred labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase.
  • Immobilization of reagents is required for certain assay methods. Immobilization entails separating the anti-pLDH antibody from any pLDH that remains free in solution. This conventionally is accomplished by either insolubilizing the anti-pLDH antibody or pLDH analogue before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al. , U.S. Patent No. 3,720,760), by covalent coupling (for e.g., using glutaraldehyde cross-linking), or by insolubilizing the anti-pLDH antibody or pLDH analog afterward, e.g. by immunoprecipitation.
  • Dose-response curves with known amounts of pLDH are prepared and compared with the tests results to quantitatively determine the amount of pLDH present in the test sample. These assays are called ELISA systems when enzymes are used as the detectable markers.
  • a conjugate of an enzyme with the pLDH is prepared and used such that when aKushalnti-pLDH antibodies binds to the pLDH, the presence of the anti-pLDH antibody modifies the enzyme activity.
  • the pLDH or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such a peroxidase. Conjugates are selected for use with anti-pLDH antibody so that binding of the anti-pLDH antibody inhibits or potentiates the enzyme activity of the label. This method per se is widely practiced under the name of EMIT.
  • Steric conjugates are used in steric hindrance methods or homogenous assays. These conjugates are synthesized by covalently linking a low-molecular-weight hapten to a small pLDH fragment so that antibody to hapten is substantially unable to bind the conjugate at the same time as the anti-pLDH antibody. Under this assay procedure, the pLDH present in the test sample will bind anti-pLDH antibody, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character of the conjugate hapten, e.g. a change in fluorescence when the hapten is a fluorophore. Sandwich assays are also useful for the determination of pLDH or anti-pLDH antibodies.
  • an immobilized anti-pLDH antibody is used to adsorb test sample pLDH, the test sample is removed as by washing, the bound pLDH is used to adsorb a second, labeled anti-pLDH antibody and bound material is then separated from residual tracer. The amount of bound tracer is directly proportional to test sample pLDH. In "simultaneous" sandwich assays, the test sample is not separated before adding the labeled anti-pLDH.
  • a sequential sandwich assay using an anti-pLDH monoclonal antibody as one antibody and a polyclonal anti-pLDH antibody as the other is useful in testing samples for pLDH.
  • a blood sample which may consist of whole blood, blood hemolysates, plasma, serum or the like, is combined with a specialized reagent.
  • the reagent includes three main components.
  • the first component is 3-acetyl pyridine adenine dinucleotide ("APAD") which functions as a coenzyme/necessary cofactor and is reduced enzymatically in the presence of pLDH.
  • APAD 3-acetyl pyridine adenine dinucleotide
  • This material is commercially available from the Sigma Chemical Corporation of St. Louis, MO.
  • the second component is a substrate which is generally defined as a molecule whose chemical conversion is catalyzed by an enzyme.
  • the substrate is a 1- lactate salt.
  • the third component of the reagent is a buffer which is used to maintain the pH of the reagent at between about 8.0- 10.0.
  • Preferred buffers include AMPSO, CHES, bis-tris propane, AMP, and TRIS but other suitable buffers capable of maintaining the proper pH may also be used..
  • the reagent and blood sample are then combined. In a preferred sample, the reagent to blood volume ratio is about 1:300 and if serum is used the preferable range is from about 1:1 to about 1:20.
  • the APAD is enzymatically reduced to APADH and the substrate is oxidized (e.g. lactate to pyruvate).
  • the APADH may thereafter be detected using spectrophotometric techniques, colorimetrically, fluorimetrically, or electrophoretically. These detection methods are well known to those of ordinary skill in the art. The foregoing are merely exemplary diagnostic assays for pLDH. Other methods now or hereafter developed that use anti-
  • LDH antibody for the determination of pLDH are included within the scope hereof, including the bioassays described above. All references cited in this specification are hereby expressly incorporated by reference. The following examples are offered by way of illustration and not by way of limitation. It should be appreciated that the Plasmodium used in each of the below examples are representative of all
  • the panel of anti-pLDH monoclonal antibodies was made by immunizing Balb-c mice to a protein fraction containing pLDH purified from red blood cells grown in vitro cultures of the P . falciparum strain D6. The authenticity of this source of pLDH was demonstrated by direct sequencing of the N-terminus. For boosting immunization, recombinant pLDH was produced by subcloning a PCR fragment amplified from genomic DNA from the P. falciparum strain D6 into the expression vector pTrc99 (Pharmacia). E.
  • Coli transformed with the resulting plasmid were subsequently induced with IPTG to produce recombinant pLDH (rpLDH).
  • Recombinant pLDH is purified from bacterial lysates over a Cibacron blue sephrose and ion exchange column chromatography.
  • Authentic pLDH was purified by Cibacron blue sepharose chromatography from culture lysates (Fig. 1).
  • pLDH activity (ADAP) can be separated from human red blood cell LDH (1st NAD peak) because pLDH binds tightly to Cibacron blue.
  • Recombinant pLDH produced in bacteria is purified the same way and shows identical biochemical characteristics to authentic pLDH.
  • Hybridoma cell lines were subcloned until monoclonality was established. Antibodies were harvested from serum free media and purified by the sequential precipitation with ammonium sulfate and caprylic acid. Antibodies were subtyped as:
  • Figure 2 shows an agarose electrophoresis gel in which active enzyme has been stained with NAD + , NBT and PES.
  • Active recombinant pLDH was used as an immunogen to raise the monoclonal antibodies, 19G7, 7G9, 17E4, 6C9a, and 6C9b.
  • microtiter plates These antibodies have been used to coat microtiter plates (see Figure 3) . These coated plates are capable of capturing enzymatically active pLDH from samples of whole blood, plasma, or lysed red blood cells.
  • the immuno-enzyme capture assay (ICpLDH) is able to capture small amounts of pLDH from blood products and allow for the unambiguous measurement of pLDH activity.
  • ICpLDH immuno-enzyme capture assay
  • MAbs captured active enzyme without adverse effects on enzyme activity.
  • the 6C9a antibody captured pLDH but at high concentrations inhibits enzyme activity. All five antibodies were subcloned and prepared and purified on a large scale.
  • sample volumes were adjusted to 150 ⁇ ls total with the addition of PBS containing 1% BSA.
  • the wells were previously coated with either the 19G7 monoclonal antibody (pan-specific) or the 17E4 monoclonal antibody (P. falciparum specific). the wells were then washed 2 times with PBS and incubated with APAD containing NBT and Diaphorase.
  • the microtiter wells are shown in Figure 6.
  • the 19G7 and 6C9a antibodies recognized a wide range of Plasmodium LDH isoforms.
  • 6C9a recognized all isoforms tested. Based on previous data on other Plasmodium enzymes, it is fully expected that the 6C9a antibody will be fully capable of recognizing the pLDH isoforms in these species as well.
  • the pLDH test was positive with 90% of the samples.
  • three of the false negative samples were from a single patient (Patient #64).
  • the samples were part of a series taken from a patient undergoing antimalarial treatment.
  • the initial sample taken was positive by ICpLDH assay, demonstrating that the reason for false negative result was not due to a lack of antibody specificity.
  • Table 2 shows the efficacy of the diagnostic test for the detection of P . vivax. Results from samples collected at Portland Veterans Administration Hospital, the London Hospital for Tropical Disease, and from a collaborative clinic in Cali Columbia were combined and tabulated.
  • ICpLDH assay On important aspect of the ICpLDH assay is apparent from inspection of several samples of patients undergoing anti malarial treatment. These data sets show that pLDH activity presents in peripheral red blood cells drops in concert with parasitemia. It is possible that plasma levels of pLDH may reflect total parasite load (sequestered and peripheral parasites) .
  • ParasightFTM antigen detection assay was found to perform comparably to the assay of the present invention using MAbs. Within a very low range of parasitemias ( ⁇ 0.001% or 50 ⁇ ls/ ⁇ l) some infected samples were found positive by pLDH but not by ParasightFTM as well as vise versa. Upon analyzing serial samples from patients undergoing anti malarial chemotherapy, the ParasightFTM assay, in contrast to the ICpLDH assay and the standard thin smear, consistently gave a positive signal even when parasites were completely cleared as judged by microscopy.
  • a nitrocellulose strip was coated with a line of 17E4 monoclonal antibody (P. falciparum specific) and a second line of 19G7 monoclonal antibody (pan specific).
  • the blood sample was then lysed with a bead conjugate made with the 6C9a antibody.
  • the lysate was then wicked up the nitrocellulase strip. If the sample contained pLDH (from P . falciparum) the bead conjugate (with bound pLDH) would bind to the 17E4 line. If the bead conjugate has pLDH from P. vivax bound, the conjugate would pass over the 17E4 line and bind only to the 19G7 line. It is anticipated that the dip stick will be able to differentiate between P. vivax and P. falciparum and is capable of detecting 5 viable parasites/ ⁇ l of blood, thus providing a solid phase "dip-stick" assay.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Méthode perfectionnée pour la détection et la différenciation des espèces participant à l'infection malarique. On a développé une série d'anticorps monoclonaux capables de capturer l'enzyme lactate-déhydrogénase et spécifiques de la LHD de plasmodium sans se lier à la LDH humaine. Les analyses d'activités et le résultat de celles-ci permet d'assurer un criblage de LDHp qui est 10 à 100 fois plus sensible que les méthodes d'analyses connues jusqu'à maintenant. L'invention concerne aussi des anticorps monoclonaux capables de distinguer les isoformes de LDH des différentes espèces de plasmodium qui infectent les humains.
PCT/US1996/020672 1995-12-29 1996-12-27 Anticorps monoclonaux et methode de capture immunologique pour la quantification et la differenciation d'especes d'hematozoaires Ceased WO1997024141A1 (fr)

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AU22404/97A AU2240497A (en) 1995-12-29 1996-12-27 Monoclonal antibodies and immuno-capture method for quantitation and speciation of malaria parasites

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049413A3 (fr) * 1999-02-19 2001-03-01 Medical Analysis Systems Inc Essai de detection rapide pour vecteurs de maladies et pathogenes vehicules par les arthropodes
KR20030088294A (ko) * 2002-05-14 2003-11-19 주식회사 에스디 신속 면역크로마토그라피법 및 효소면역측정법에 의해사람 전혈에서 말라리아 항원 젖산탈수소효소를 검출하는방법 및 진단 키트
WO2015069965A1 (fr) * 2013-11-08 2015-05-14 Veterinary Diagnostics Institute, Inc. Procédé et appareil pour détecter des maladies à transmission vectorielle chez des mammifères
CN106908602A (zh) * 2017-01-06 2017-06-30 苏州万木春生物技术有限公司 一种疟原虫pf/pan检测试纸的制备方法
EP2729809B1 (fr) * 2011-07-07 2018-10-17 DuPont Nutrition Biosciences ApS Analyse
CN115785275A (zh) * 2021-09-10 2023-03-14 东莞市朋志生物科技有限公司 一种抗疟原虫的抗体及其应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049413A3 (fr) * 1999-02-19 2001-03-01 Medical Analysis Systems Inc Essai de detection rapide pour vecteurs de maladies et pathogenes vehicules par les arthropodes
KR20030088294A (ko) * 2002-05-14 2003-11-19 주식회사 에스디 신속 면역크로마토그라피법 및 효소면역측정법에 의해사람 전혈에서 말라리아 항원 젖산탈수소효소를 검출하는방법 및 진단 키트
EP2729809B1 (fr) * 2011-07-07 2018-10-17 DuPont Nutrition Biosciences ApS Analyse
WO2015069965A1 (fr) * 2013-11-08 2015-05-14 Veterinary Diagnostics Institute, Inc. Procédé et appareil pour détecter des maladies à transmission vectorielle chez des mammifères
CN106908602A (zh) * 2017-01-06 2017-06-30 苏州万木春生物技术有限公司 一种疟原虫pf/pan检测试纸的制备方法
CN115785275A (zh) * 2021-09-10 2023-03-14 东莞市朋志生物科技有限公司 一种抗疟原虫的抗体及其应用
WO2023035974A1 (fr) * 2021-09-10 2023-03-16 东莞市朋志生物科技有限公司 Anticorps contre le plasmodium et son application
CN115785275B (zh) * 2021-09-10 2023-09-22 东莞市朋志生物科技有限公司 一种抗疟原虫的抗体及其应用

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