WO1986000646A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation Download PDF

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Publication number
WO1986000646A1
WO1986000646A1 PCT/GB1985/000296 GB8500296W WO8600646A1 WO 1986000646 A1 WO1986000646 A1 WO 1986000646A1 GB 8500296 W GB8500296 W GB 8500296W WO 8600646 A1 WO8600646 A1 WO 8600646A1
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shigella
antigen
antibody
specific
monoclonal antibody
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PCT/GB1985/000296
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English (en)
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Bruce William Wright
Peter John Cox
Alice Margaret Noyes
Danny Widdows
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Technology Licence Company Limited
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Publication of WO1986000646A1 publication Critical patent/WO1986000646A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • MONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use.
  • Shigella is described in Zinsser Microbiology (17th ed.) 739-741, as primarily human pathogens, but they are also isolated occasionally from other animals.
  • the genus Shigella is classified in the tribe Escherichieae, and Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei constitute the four species of the genus. Speciation is based upon serologic and biochemical reactions.
  • Shigella species have been among the Shigella species on the basis of antigenic structures.
  • the Shigellae may be divided into four major 0 antigenic groups designated A, B, C and D, which correspond to the species S_. dysenteriae, S_. flexneri, S_. boydii and S_. sonnei, respectively.
  • Each major group or species is also sub-divided into types on the basis of the 0 antigen. These sub-groups are designated by arabic numbers.
  • Bacterial diarrhea is a common and often serious condition manifest as fluid loss from the bowel, leading in many cases to dehydration, and occasionally death.
  • Shigella In addition to diarrhea, Shigella is known to cause gram-negative sepsis which is a bloodstream infection. It is one of the major infectious disease problems encountered in modern medical centres. While it can be transient and self-limited, severe gram-negative sepsis constitutes a medical emergency. Present treatment and diagnosis of Shigella infections vary depending on the locus of the infection. It is estimated that in the United States and Europe many millions of cases of bacterial diarrhea occur annually, of which several million are seen by a physician or admitted to a hospital. Because of the self-limiting nature of the adult disease, most people do not seek treatment. Of the people seeking treatment, bacterial diagnosis of diarrhea is presently made by stool culture techniques. These techniques are generally performed only in hospitals and are slow, requiring one to three days. During this time, the patient is exposed, if treated, to the expense and potential hazard of inappropriate therapy. However, if not treated, the patient is exposed to the hazard of a deteriorating condition pending the test result and initiation of therapy.
  • the test for gram-negative sepsis involves processing blood and urine cultures and other procedures on occasion.
  • blood culture tests are cumbersome. They require a day, and often several days, to return results. They require expert laboratory skills because of the complex nature of human blood which tends to interact non-specifically with many of the test reagents.
  • existing methods of detection of Shigella with high accuracy in diarrheal or gram-negative sepsis are less than satisfactory in that they consume large amounts of expensive skilled labour and laboratory time, generally taking one and often several days before returning results.
  • the present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Shigella antigens and/or organisms.
  • the present invention comprises monoclonal antibodies specific for an antigen of Shigella; in particular, the 0A1, OA2, OA3, OA4, OA5, OA6, OA7, OA8, OA9 and OA10 antigens of Shigella dysenteriae, the OBl,.OB2, OB3, OB4, OB5 and OB6 antigens of Shigella flexneri, the OCl, OC2, OC3, OC4, OC5, OC6, OC7, OC8, OC9, OC10, OCll, OC12, OC13, OC14 and OC15 antigens of Shigella boydii, and the ODl antigen of Shigella sonnei, the invasiveness antigens 1 and 2, attachment antigens 1 and 2, enterotoxin 1 and 2, as well as a monoclon
  • the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Shigella antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Shigella or to a particular species thereof and having linked thereto an appropriate label.
  • the label can be, for example,, a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom or particle.
  • the invention further comprises the process for diagnosing the presence of Shigella antigens or organisms in a specimen, comprising contacting said specimen with the labelled monoclonal antibody in an appropriate immunoassay procedure.
  • the invention is also directed to a therapeutic composition
  • a therapeutic composition comprising a monoclonal antibody for an antigen of Shigella and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Shigella.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Shigella antigen, with an appropriate myeloma cell line, preferably NSO (uncloned) , P3NS1-Ag4/1, or Sp2/0 Agl4.
  • the resultant product is then cultured in a standard HAT (hypoxanthine, aminopterin and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilising immunoassay techniques which will be described below.
  • the immunised spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e. mice, rats and rabbits) , bovines, ovines and canines, but the present invention will be described in connection with mice.
  • the mouse is first immunised by injection of the particular Shigella antigen chosen, generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the appropriate Shigella antigen, and then killed so that the immunised spleen may be removed. The fusion can then be carried out utilising immunised spleen cells and an appropriate myeloma cell line.
  • the fused cells yielding an antibody which gives a positive response to the presence of the particular Shigella antigen are removed and cloned utilising any of the standard methods.
  • the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Shigella antigen.
  • the monoclonal antibody selected, which is specific for the particular Shigella antigen or species, is then bound to an appropriate label.
  • Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture in vivo in mammals such as mice.
  • the monoclonal antibodies may be labelled with various labels, as exemplified above.
  • the present invention will be described with reference to the use of an enzyme-labelled monoclonal antibody. Examples of enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase and urease.
  • Such linkage with enzymes can be accomplished by any known method, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
  • testing is carried out employing one of a wide variety of conventional immunoassay methods. The particular method chosen will vary according to the monoclonal antibody and the label chosen.
  • enzyme immunoassays are preferred owing to their low cost, reagent stability, safety, sensitivity and ease of procedure.
  • EIA enzyme-linked immunosorbent assay
  • EIA is a solid-phase assay system which is similar in design to the radiometric assay, but which utilises an enzyme in place of a radioactive isotope as the immunoglobin marker.
  • Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluorescent probes. A non-labelled antigen and a specific antibody are combined with identical fluorescently-labelled antigen. Both labelled and unlabelled antigen compete for antibody binding sites. The amount of labelled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of non-labelled antigen. Examples of this particular type of fluorescent-immunoassay include heterogeneous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate-Labelled Fluorescent Immunoassay. The most suitable fluorescent probe, and the one most widely used, is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimised for the probe utilised in the particular assay, and in which the effect of scattering can be minimised.
  • Fluorescence polarisation In fluorescence polarisation, a labelled sample is excited with polarised light and the degree of polarisation of the emitted light is measured. As the antigen binds to the antibody, its rotation slows down and the degree of polarisation increases. Fluorescence polarisation is simple, quick and precise. However, at the present time, its sensitivity is limited to the micromole per litre range and upper nanomole per litre range with respect to antigens in biological samples.
  • Luminescence is the emission of light by an atom or molecule as an electron is transferred to the ground state from a higher energy state.
  • the free energy of a chemical reaction provides the energy required to produce an intermediate reaction or product in an electronically-excited state. Subsequent decay back to the ground state is accompanied by emission of light.
  • - Bioluminescence is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction. The best known chemiluminescent substance is luminol.
  • a further aspect of the present invention is a therapeutic composition
  • a therapeutic composition comprising one or more of the monoclonal antibodies to the particular Shigella antigen or species, as well as a pharmacologically-acceptable carrier or diluent.
  • Such compositions can be used to treat humans and/or animals afflicted with some form of shigellosis and they are used in amounts effective to cure; the amount may vary widely, depending upon the individual being treated and the severity of the infection.
  • One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens or species of Shigella in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Shigella alone or as part of a kit i containing antibodies that can identify other bacterial genera or species of Shigella and/or other bacteria.
  • conjugated or labelled monoclonal antibodies for antigens and/or species of Shigella and other gram-negative bacteria can be utilised in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible
  • the monoclonal test is an advance over existing procedures in-that it is more accurate than existing tests; it gives "same day” results, provides convenience to the patient and improves therapy as a result of early, accurate diagnosis; and it reduces labour costs and laboratory time required for administration of the tests.
  • the kit may be sold individually or included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Shigella.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Shigella antigen or species, as well as any appropriate stains. counterstains or reagents. Further embodiments include kits containing at least one control sample of a Shigella antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Shigella organisms in a particular sample.
  • Monoclonal diagnostics which detect the presence of Shigella antigens can also be used in periodic testing of water sources, food supplies and food processing operations.
  • the present invention describes the use of the labelled monoclonal antibodies to determine the presence of a standard antigen
  • the invention can have many applications in diagnosing the presence of antigens by determining whether specimens, such as urine, blood, stool, water and milk, contain the particular Shigella antigen. More particularly, the invention could be utilised as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.
  • the invention will be further illustrated in connection with the following Examples which are set forth for purposes of illustration only and not by way of limitation.
  • the monoclonal antibodies of the present invention were prepared generally according to the method of Kohler and Milstein, supra.
  • API Analytical Profile Index (ref. Ayerst Laboratories)
  • DMEM Dulbeccos Modified Eagles Medium
  • FCS Foetal Calf Serum
  • % T refers to vaccine concentrations measured in a 1 cm light path
  • Shigella boydii bearing the antigen OC1 was obtained from the National Collection of Type Cultures (NCTC accession No. 9731) and tested against standard reference typing sera to confirm its typing. More specifically, the Shigella boydii was removed from the lyophile, grown on blood agar, and tested by conventional biochemical (API) and agglutination tests with appropriate antisera to confirm it identity and purity. The cells were then transferred to DMEM, grown, and harvested for use as a source of antigen. The organisms were washed in phenol saline by repeated centrifugation and were resuspended in phenol saline. 3. Animal Immunisatio si Balb/c mice were injected with the prepared antigen.
  • NCTC accession No. 9731 National Collection of Type Cultures
  • mice were given one intraperitoneal injection per week for three weeks (0.05 ml 80% T vaccine) followed by six intravenous injections every other week of LDlO of boiled killed Shigella boydii 0C1 vaccine prepared as above for a total of eleven weeks.
  • the mice were bled approximately six days after the last injection and the serum tested for antibodies by assay.
  • the conventional assay used for this serum titer testing was the enzyme-linked immunosorbent assay system.
  • a positive titer of at least 10,000 a mouse was selected as a fusion donor and given a booster injection of 80% T vaccine intravenously, three days prior to splenectomy.
  • the selected donor mouse was killed and surface sterilised by immersion in 70% ethyl alcohol.
  • the spleen was then removed and immersed in approximately 2.5 ml of DMEM to which had been added 3% FCS.
  • the spleen was then gently homogenised in a LUX homogenising tube until all cells had been released from the membrane and the cells were washed in 5 ml 3% FCS DMEM.
  • the cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris was then rewashed in 5 ml 3% FCS DMEM. 50 ml of suspension were then made in 3% FCS DMEM.
  • the myeloma cell line used was NSO (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using a tissue culture medium DMEM containing 3% FCS.
  • the spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 6.00 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS DMEM.
  • 0.1 ml of the suspension was diluted to 1 ml and a haemacytometer with phase microscope was used.
  • 0.1 ml of the suspension was diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope were used to count the stained nuclei of the cells.
  • the resultant cell pellet was placed in a 37°C water bath. Over the period of one minute, 1 ml of a 50% w/v solution of polyethylene glycol 1500 (PEG) in saline
  • the wells were kept undisturbed and cultured at 37°C in 9% C0 2 -air at approximately 100% humidity.
  • the wells were analysed for growth utilising the conventional inverted microscope procedure, after about 5 to 10 days.
  • screening tests for the specific monoclonal antibody were made utilising the conventional enzyme immunoassay screening method described, below- Somewhere around 10 days to 14 days after fusion, sufficient antibody against the Shigella boydii OC1 antigen was developed in at least one well.
  • a freshly-prepared stock solution of sterile 1.2% agar in double-distilled water with an equal volume of double-strength DMEM and additives was maintained at 45"C.
  • This solution (10 ml) was then aliquoted into 10 cm Petri dishes, to form a base layer.
  • An overlay of equal volumes of agar and cells in 18% FCS-DMEM was spread evenly over the base. The cells were allowed to multiply for approximately 10 days at 37°C, 7-9% CO_, 9.5% RH. Viable separate colonies were picked off the agar surface and placed into 60 wells of a 96-well microtitre tray in 18% FCS-DMEM. After a further period of growth, the supernatants were assayed for specific antibody by the standard enzyme immunosorbent assay.
  • the monoclonal antibodies from the clones were screened by the standard techniques for binding to Shigella boydii NCTC 9731 prepared as in the immunisation, and for specificity in a tes battery of Shigella species and related genera bearing different antigens. Specifically, a grid of microtiter plates- containing a representative selection of O-serotype organisms, i.e. Pseudomonas, Klebsiella, Serratia and Enterobacter, was prepared, boiled, and utilised as a template to define the specificity of the parent
  • mice were primed with pristane for at least 7
  • the fluid was titrated, as noted above, to establish presence and level of antibody, and purified. Purification is accomplished using the Protein A-Sepharose method, in which about 10 ml of the ascites fluid are filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then diluted with twice its own volume of cold phosphate buffer (0.1 M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose which had previously been equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer.
  • cold phosphate buffer 0.1 M sodium phosphate, pH 8.2
  • the monoclonal antibody was eluted with citrate buffer (0.1 M sodium citrate, pH 3.5) into sufficient 1M Tris buffer, pH 9.0, to raise the pH immediately to about 7.5.
  • citrate buffer 0.1 M sodium citrate, pH 3.5
  • 1M Tris buffer pH 9.0
  • the eluate was dialysed in 2 x 1000 ml PBS, pH 7.4 at +4°C, and stored at -20°C.
  • Monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml of PBS pH 7.4, at +4°C. After dialysis, the volume was made up to 2.5 ml with PBS and 25 ul of a 20% solution of glutaraldehyde in PBS was added. The conjugation mixture was left at room temperature for 1.5 hours.
  • the enzyme immunoassay method was used for testing. This assay method comprises coating the wells of a standard polyvinyl chloride microtiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenol phosphate.
  • the monoclonal antibody was found to be specific for the OC1 antigen of Shigella boydii.
  • the monoclonal antibody was also tested and shown to be of the Class IgG3.
  • the particular epitopic site to which the antibody attaches to the antigen can also be determined.
  • the same enzyme immunoassay method can also be used to determine whether diagnostic specimens such as urine, blood, stool, water or milk contain the antigen. In such cases, the antibody can first be bound to the plate. Examples 2 to 11
  • Example 1 The procedure of Example 1 was followed in each of 10 cases, with differences outlined below, to prepare monoclonal antibody conjugates for various antigens of the genus Shigella.
  • Examples 2 to 7 Shigellae boydii bearing the respective antigens OC4, OC6, OC7, OC10 and OCll (twice) were used; in Example 8, Shigella flexneri bearing the OBI antigen; in Examples 9 and 10, Shigellae dysenteriae bearing the OAl and OA10 antigens; and, in Example 11, Shigella sonnei bearing the OD 1 antigen. These were all obtained from NCTC where the respective accession numbers are 9330, 9332, 9333, 9357, 9321, title 3, 4837, 9351 and 9774.
  • mice were vaccinated intraperitoneally with the prepared antigen (0.05 ml of 80% T vaccine) followed by an intravenous boost after six weeks.
  • the mice showed a suitably elevated titre to the immunising antigen, they were used as a source of spleen cells for fusion.
  • mice were immunised with a dose of 0.05 ml of 80% T vaccine given in one intraperitoneal injection per week for three weeks followed by one intravenous injection per month, for three months, of boiled, killed Shigella boydii OC6 prepared as in Example 1.
  • the mice were immunised with a dose of 0.5 ml of 80% T vaccine given in one intraperitoneal injection per week for three weeks, followed by intravenous injection per week for two weeks, of boiled and killed S_. boydii prepared as in Example 1, for a total of five weeks.
  • mice were immunised with a dose of 0.05 ml of 80% T vaccine given in one intraperitoneal injection per week for three weeks, followed by an intravenous injection per week for three weeks, and then four intravenous injections every other week, of boiled, killed Shigella dysenteriae, for a total of 14 weeks.
  • Example 2 In the antigen preparation step for Examples 2, 3 and 5 to 10, the organisms were boiled as well as washed, in phenol saline. In Examples 4 and 11, the organisms were boiled and washed in saline. In Example 11, resuspension was in phenol saline.
  • the agar method alone was used; in Examples 3 and 5, the limiting dilution and agar methods; and, in Examples 4 and 11, the limiting dilution method (twice).
  • the monoclonal selection step Shigella and E. coli were invariably used, Pseudomonas in all except Examples 3 and 7, Klebsiella in all except Examples 2, 3, 7 and 8, Salmonella in all except Example 4, and Serratia in Example 2 only.
  • the SP-Sephadex was allowed to settle and the supernatant decanted.
  • the SP-Sephadex was packed in a column, washed with 60 ml of 0.1M acetate buffer, pH 5.0, and eluted with 60 ml of the same buffer plus 1M sodium chloride.
  • the eluate was stirred at +4°C, and an equal volume of saturated ammonium sulphate added slowly.
  • the suspension was stirred for a further 30 min, and then the precipitate was harvested by centrifugation at 10,000 g for 10 min.
  • the precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) .
  • the dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 min and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer.
  • the monoclonal antibody was eluted with phosphate/EDTA buffer.
  • the antibody purification step for Examples 6, 8, 9 and 10 was accomplished using the ammonium sulphate precipitation/DEAE-cellulose method. Ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 min. The ascites was then stirred at +4°C and an equal volume of cold, saturated ammonium sulphate added slowly. The mixture was stirred for a further 30 min after the addition was complete. The precipitate was harvested by centrifugation at 10,000 g for 10 min. The precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, +- 0.02% sodium azide) . The.
  • the antibody purification step for Example 11 involved the supernatant on Protein A-Sepharose method. To 1 litre of culture supernatant were added 100 ml of 1.0M Tris buffer, pH 8.2. The Tris buffered supernatant was applied at a flow rate of 1 ml/min to a 1 ml column of Protein A-Sepharose, previously equilibrated with 0.1M Tris buffer, pH 8.2. The column was then washed with 40 ml of 0.1M Tris buffer. The monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient IM Tris buffer, pH 9.0, to raise the pH immediately to about 7.5. The eluate was dialysed in PBS, pH 7.4, at 4°C, and stored at -20°C.
  • citrate buffer 0.1M sodium citrate, pH 3.5
  • the benzoquinone method was used. 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25M sodium phosphate buffer, pH 6.0, at +4°C. Para-benzoquinone, 18 mg, was dissolved in warm AR ethanol, 0.6 ml, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour.
  • alkaline phosphatase Sigma Type VII-T
  • the conjugate was dialysed against 2 x 1000 ml of PBS + 0.02% sodium azide at +4°C. An equal volume of glycerol was added. The conjugate was sterile- filtered through a 0.22 ⁇ m membrane filter into a sterile amber vial and stored at +4°C.
  • Example 12 The monoclonal antibodies were also tested and shown to be of the classes IgG2a (Examples 2, 5 and 6), IgG3 (Examples 3, 9, 10 and 11), IgGl (Examples 4 and 7) and IgM (Example 8) .
  • IgG2a Examples 2, 5 and 6
  • IgG3 Examples 3, 9, 10 and 11
  • IgGl Examples 4 and 7
  • IgM Example 8
  • Example 1 The same procedure as in Example 1 may be utilized in preparing a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Shigella, but using another Shigella obtained from the National Collection of Type Cultures.
  • Tests using the present invention are superior to the existing tests based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expense; and (v) improved therapy based upon early, precise diagnosis.

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Abstract

Anticorps monoclonaux du genre Shigella, anticorps étiquetés, compositions et kits les contenant, et leur utilisation dans le diagnostic d'antigènes et dans le traitement.
PCT/GB1985/000296 1984-07-03 1985-07-02 Anticorps monoclonaux et leur utilisation WO1986000646A1 (fr)

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GB848416845A GB8416845D0 (en) 1984-07-03 1984-07-03 Monoclonal antibodies

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Publication number Priority date Publication date Assignee Title
WO1987003205A1 (fr) * 1985-11-22 1987-06-04 Coral Sociedade Brasileira De Pesquisas E Desenvol Anticorps couples a une enzyme
WO1999055364A2 (fr) * 1998-04-24 1999-11-04 Henry M. Jackson Foundation For The Advancement Of Military Medicine Proteines provoquant la secretion de proteines virulentes et vaccins contenant lesdites proteines
CN107064488A (zh) * 2017-05-02 2017-08-18 中国农业科学院兰州兽医研究所 一种猪瘟病毒血清总抗体固相阻断elisa试剂盒所用抗原的制备方法

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Title
Biological Abstracts, Volume 77, Nr. 7, 1984, Philadelphia, PA, (US) N.I.A. CARLIN et al.: "Moncclonal Antibodies Specific for O-Artigenic Polysaccharides of Shigella Flexneri: Clones Binding to II, II:3,4 and 7,8 Epitopes", see page 5645, column 2, Abstract Number 51448 J Clin. Microbiol., 1983, 1183-1189 *
CHEMICAL ABSTRACTS, Volume 100, Nr. 5, 30 January 1984, Columbus, Ohio, (US) D.R. Bunixe et al.: "Synthesis of Shigella Flexneri O-Antigenic Repeating Units. Conformational Probes and Aids to Monoclonal Antibody Production", see page 332, column 1, Abstract Number 33054g, & ACS Symp. Ser. 1983, 49-63 (Eng) *
CHEMICAL ABSTRACTS, Volume 102, Nr. 15, 15 April 1985, Columbus, Ohio, (US) A. DONOHUE-ROLFE et al.: "Pathogenesin of Shigella Diarrhea. IX Simplified High-Yeald Purification of Shigella Toxin and Characterization of Subunit Composition and Function by the use of Subunit-Specific Monoclonal and Polyclonal Antibodies", see page 192, column 2, Abstract Number 126945e & J.Exp.MED., 1984, 1767-1781 (Eng) *
CHEMICAL ABSTRACTS, Volume 99, Nr. 5, 1 August 1983, Columbus, Ohio, (US) D.E. GRIFFIN et al.: "Isolation and Characterization of Monoclonal Antibodies to Shiga Toxin", see page 401, column 2, Abstract Number 36914j & Infect. Immun., 1983, 430-433 (Eng) *

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WO1987003205A1 (fr) * 1985-11-22 1987-06-04 Coral Sociedade Brasileira De Pesquisas E Desenvol Anticorps couples a une enzyme
WO1999055364A2 (fr) * 1998-04-24 1999-11-04 Henry M. Jackson Foundation For The Advancement Of Military Medicine Proteines provoquant la secretion de proteines virulentes et vaccins contenant lesdites proteines
WO1999055364A3 (fr) * 1998-04-24 2000-01-06 Jackson H M Found Military Med Proteines provoquant la secretion de proteines virulentes et vaccins contenant lesdites proteines
US6342352B1 (en) 1998-04-24 2002-01-29 The Henry M. Jackson Foundation For The Advancement Of Military Medicine Method of detecting shigella and shigella mxiM DNA
CN107064488A (zh) * 2017-05-02 2017-08-18 中国农业科学院兰州兽医研究所 一种猪瘟病毒血清总抗体固相阻断elisa试剂盒所用抗原的制备方法

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