WO1986001806A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation Download PDF

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WO1986001806A1
WO1986001806A1 PCT/GB1985/000408 GB8500408W WO8601806A1 WO 1986001806 A1 WO1986001806 A1 WO 1986001806A1 GB 8500408 W GB8500408 W GB 8500408W WO 8601806 A1 WO8601806 A1 WO 8601806A1
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antibody
antigen
monoclonal antibody
specific
immunoassay
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PCT/GB1985/000408
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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 WO1986001806A1 publication Critical patent/WO1986001806A1/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
    • C07K16/1232—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 from Escherichia (G)
    • 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.
  • IS coli is described in Zinsser Microbiology (17th ed.) 734-5.
  • capsular antigens designated Kl to K99
  • flagellar antigens designated Hi to H42
  • enterotoxins i.e. heat-labile (LT) , heat-stable (ST) and vero toxin (VT)
  • binding antigens divided into two major factions, 1 and 2
  • endotoxins produced by 33. coli and vero toxins which attack vero cells.
  • IS. coli occupies a unigue position among opportunistic enteric bacilli in that certain strains are capable of causing primary intestinal disease as well as extra-intestinal infection.
  • IS. coli has been the subject of more experimental research than any other micro-organism, especially in the field of molecular biology.
  • IS. coli is the most common cause of urinary tract infection in man. It is also the most frequent cause of gram-negative sepsis and has been isolated from pneumonia, wounds and cerebrospinal fluid. 13. coli is a major cause of neonatal meningitis but it is rarely seen in older populations. The mortality in IS. coli meningitis is between 40 and 80% in new-borns, , and in survivors the majority have subsequent neurologic or developmental abnormalities. The invasiveness of E_. coli in this disease is apparently due to the capsular antigen K-l. —• col has also been associated with gastrointestinal disease in both man and animals. The enterpathogenicity of 33.
  • E coli appears to be mediated, either by production of an enterotoxin or by a shigella-like penetration of the intestinal mucosa.
  • the toxin causes fluid accumulation in the jejunal and ileal portion of the intestine, while the E. coli that causes intestinal mucosal penetration resides primarily in the colon.
  • Present treatment and diagnosis of E . coli 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.
  • 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.
  • urinary tract infections a microscopic examination is made, to determine the presence of micro-organisms as a preliminary screening. The microscopic examination cannot distinguish among the gram-negative bacteria.
  • a second step is a urine culture to identify the organism isolated in the urine sample. A delay in diagnosis and initiation of treatment can result in serious complications.
  • the present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of E. coli antigens and/or organisms.
  • the present invention comprises monoclonal antibodies specific for an antigen of IS. coli; in particular, the antigen or species of E. coli; the 01 to 0159 (inclusive) antigen or antigens of E_. coli; the Kl to K99 (inclusive) antigen or antigens of IS. coli; the Hi to H42 (inclusive) antigens of IS. coli; the binding antigens 1 and 2 of E. coli; the enterotoxins LTl of E. coli; the enterotoxins ST1 and ST2 of E_. coli; the endotoxins of E. coli; as well as a monoclonal antibody broadly cross-reactive with an antigen for each species (or substantially all species) of the genus E. coli.
  • the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Escherichia antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Escherichia 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 Escherichia 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 Escherichia and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Escherichia.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Escherichia 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 Escherichia antigen chosen, e.g for a period of approximately eleven weeks.
  • the mouse When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the appropriate Escherichia 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
  • Escherichia 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 Escherichia antigen.
  • the monoclonal antibody selected, which is specific for the particular Escherichia 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.
  • EIA enzyme-linked immunosorbent assay
  • 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.
  • 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 Escherichia 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 Escherichia infection 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 Escherichia in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Escherichia alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Escherichia and/or other bacteria. in the past, there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens; e.g. urine with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis.
  • kits could be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis.
  • conjugated or labelled monoclonal antibodies for antigens, and/or species of Escherichia 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 Escherichia or other gram-negative sepsis.
  • 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 Escherichia.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Escherichia antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Escherichia antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Escherichia organisms in a particular sample.
  • Monoclonal diagnostics which detect the presence of Escherichia 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 Escherichia 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
  • PBS Phosphate Buffered Saline
  • Escherichia coli bearing an 08 antigen was obtained from the National Collection of Type Cultures (NCTC accession No. 9008) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) .
  • the Escherichia coli was removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity.
  • the bacteria were transferred for growth on to TSB and harvested. The organisms were boiled and washed in saline by repeated centrifugation, and then resuspended in formol saline.
  • mice Six Balb/c mice were injected with the prepared antigen, over five weeks. They were given one intraperitoneal injection per week for three weeks (0.1 ml 80% T vaccine) , followed by three intravenous injections, each after one week intervals, of LD- n of boiled killed Escherichia coli 08 antigen prepared as above. 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.
  • mice When the mice showed antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection (0.02 ml 80% T vaccine) intravenously, three days prior to splenectomy.
  • a booster injection 0.02 ml 80% T vaccine
  • Spleen cells from the immune mice were harvested three days after boosting, by conventional techniques.
  • the donor mouse selected was killed and surface-sterilised by immersion in 70% ethyl alcohol.
  • the spleen was then removed and immersed in approximately 2.5 ml 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 suspension were then made in 3% FCS-DMEM.
  • the myeloma cell line used was NS0 (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, as 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 600 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. 1 ml of a 50 w/v solution of polyethylene glycol 1500 (PEG) in saline Hepes, pH approximately 7.5, was added, and the mixture gently stirred for approximately 1.5 minutes. 10 ml serum-free tissue culture medium DMEM were then slowly added, followed by up to 50 ml of such culture medium, centrifugation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight
  • 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.
  • the clones were assayed by the enzyme immunoassay method to determine antibody production, and a positive clone recloned using the standard agar method.
  • agar method 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., 95% RH.
  • the monoclonal antibodies from the clones were screened by the standard techniques for binding to Escherichia coli NCTC 9008, prepared as in the immunisation, and for specificity in a test battery of Escherichia coli and related species bearing different antigens. Specifically, a grid of microtitre plates containing a representative selective of organisms was prepared, boiled, and utilised as a template to define the specificity of the parent group. The EIA immunoassay noted above may be used.
  • the monoclonals had the appropriate specificity (to the 08 antigen) , and were negative with respect to other E_. coli, Shigella, Salmonella and Serratia.
  • mice were primed with pristane for at least 7
  • ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes. 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 " minutes after the addition was complete. The precipitate was harvested by centrifugation at 10,000 g for 10 minutes. 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 solution was dialysed vs 2 x 1000 ml of the same buffer at +4 C. The dialysed, redissolved precipitate was centrifuged at
  • Monoclonal antibody was dialysed with alkaline phosphatase .(Sigma Type VII-T) , against 2 x 1000 ml of phosphate buffered saline (PBS), pH 7.4 at +4 C. After dialysis the volume was made up to 2.5 ml with PBS and 25 ⁇ l of a 20% glutaraldehyde in PBS solution added. The conjugation mixture was left at room temperature for 1.5 hours.
  • the enzyme immunoassay method was used for testing. This method comprises coating the wells of a standard polyvinyl chloride (PVC) microtitre -tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenyl phosphate.
  • PVC polyvinyl chloride
  • the monoclonal antibodies were found to be specific for the 08 antigen of Escherichia coli.
  • the monoclonal antibodies were also tested and shown to be of the Class IgG2b.
  • 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 42
  • Example 1 The procedure as in Example 1 was followed in each of 4i cases, with differences outlined below, to prepare monoclonal antibodies and conjugates for various antigens of the genus Escherichia coli.
  • NCTC 9001 (01) , NCTC 11100 (02) , NCTC 9004 (04) , NCTC 9005 (05) , NCTC 11105 (06) , NCTC 9007 (07) , NCTC 9003 (03) , SSI A84a (09) , NCTC 9010 (010) , NCTC 9011 (011) , NCTC 9013 (013) , NCTC 9014 (014) , NCTC 9015 (015) , NCTC 9017 (017) , PHLS E20697/o (018) , NCTC 9020 (020) , NCTC 9021 (021) , NCTC 10249 (025) , NCTC 9027 (027) , NCTC 9028 (028), SSI 6181-66 (073) , NCTC 9078 (078) , CVL B41 (0101) , PHLS E482/o (0136) , PHLS E27048
  • the growth medium was DMEM in Examples 2, 7, 8, 9, 11, 15,, 16, 19, 20, 22, 24, 25, 26, 27, 28, 34, 35, 36, 37 and 39.
  • the organisms were washed in saline in Examples 4, 7, 9, 10,22, 24, 36 and 39. Washing with saline was used in Examples 14, 18 and 21, boiling and washing with phenol saline in Examples 16, 19 and 25, and boiling and washing with formol saline in Example 17.
  • Example 2 In Examples 2, ' 8, 14, 15, 16, 19, 20, 25, 26, 27, 28, 34 and 35, resuspension was in phenol saline.
  • the antigens used in Examples 30, 31, 32, 33, 37, 40, 41 and 42 were supplied in pure form.
  • the animal immunisation procedure of Example 1 was modified by the addition of a further iv injection, after a further 25 weeks (Examples 2 and 39) , 16 weeks (Example 4) , 20 weeks (Example 8) , 3 weeks (Examples 32 and 34) or 8 weeks (Example 35) .
  • Intrasplenic preparation was used in Examples 16, 25, 26, 27 and 28.
  • injections and intervals were as follows: the sequence in Example 3 was ip-l-ip-l-ip-2-iv-2-iv-5-iv-4-iv-2-iv-2-iv- 2-iv-2-iv-2-iv; the sequence in Example 6 was ip-l-ip-l-ip-l-iv-8-iv-2-iv-l-iv; the sequence in Example 7 was im (in CFA)-6-iv(in PBS) ; the sequence in Example 9 was ip-l-ip-l-ip-l-iv-2-iv-l-iv-3 days-iv; the sequence in Example 10 was ip-l-ip-2-ip-l-iv-l-iv-l-iv- 12-iv-2-iv; the sequence in Example 11 was im (in CFA)-9-iv(in PBS) ; the sequence in Example 14 was ip-l-ip
  • Example 40 and 41 was ip-l-ip-l-ip-2-iv-4-iv; and the sequence in Example 42 was ip-l-ip-l-ip-l-iv-2-iv-l-iv-3-iv.
  • Example 30 7 8 x 10 in Example 30.
  • 2 x 10 spleen cells were used in Example 3, 1.4 x 10 in Examples, 17, 22, 34 and 35, 1.5 8 7 x 10 in Examples 24, 40 and 41, 5.4 x 10 in Example 30, and 1.44 x 10 in Example 42.
  • DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume. The culture was then allowed to overgrow, to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected.
  • ascites fluid was 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 (O.lM sodium phosphate, pH 8.2).
  • the diluted ascites was applied to a 2 ml column of Protein A-Sepharose, previously equilibrated with phosphate buffer.
  • the column was washed with 40 ml of phosphate buffer.
  • the monoclonal antibody was eluted with citrate buffer (O.lM 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.
  • the suspension was stirred for a further 30 minutes, and then the precipitate was harvested by centrifugation at 10,000 g for 10 minutes.
  • 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 minutes 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.
  • 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 O.lM TRIS buffer, pH 8.2. The column was then washed with 40 ml of O.lM TRIS buffer.
  • the monoclonal antibody was eluted with citrate buffer (O.lM 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.
  • the antibody purification step for Examples 10, 16, 26 and 28 was conducted as follows:
  • 500 ml tissue culture supernatant were concentrated ' to about 50 ml on an Amicon XM300 ultrafiltration membrane.
  • the concentrate was diluted to 500 ml with 0.9% sodium chloride and reconcentrated to about 50 ml.
  • the concentrate was stirred at +4 C, and an equal volume of saturated ammonium sulphate added slowly. The suspension was stirred for a further 30 minutes. The precipitate was then harvested by centrifugation at
  • TRIS/acetic acid buffer O.lM TRIS/acetic acid + 0.02% sodium azide, pH 7.5
  • the redissolved, dialysed precipitate was centrifuged at 30,000 g for 30 minutes, filtered through a 0.45 ⁇ m membrane filter and applied to a 215 mm x 300 mm TSK G 3000SW gel filtration column.
  • the monoclonal antibody was eluted with TRIS/acetic acid buffer and concentrated by vacuum dialysis.
  • the antibody purification step for Examples 21, 27 and 29 was conducted as follows:
  • Ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes.
  • the ascites was diluted with 9 times its own volume of cold PBS and stirred at -4 C.
  • An equal volume of cold, saturated ammonium sulphate was added slowly.
  • the mixture was stirred for a further 30 minutes after addition was complete.
  • the precipitate was harvested by centrifugation at 10,000 g for 10 minutes.
  • the precipitate was dissolved in a minimum volume of cold TRIS-acetate buffer (O.lM TRIS pH 7.5 with glacial acetic acid + 0.02% sodium azide).
  • the solution was dialysed versus 2 x 1000 ml of the same buffer at +4 C.
  • the dialysed, redissolved precipitate (5.4 ml) was centrifuged at 30,000 g for 20 minutes ' then filtered through a 0.45 ⁇ m membrane filter. A portion of the filtrate (1.0 ml) was applied to a 21.5 mm x 300 mm TSK
  • Antibody conjugation in Examples 10, 17, 21, 25, 26, 28, 34 and 42 was by the benzoquinone method.
  • 24 mg 5 alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 300 ml of 0.25 M sodium phosphate buffer, pH 6.0, at +4 C.
  • 18 mg p-benzoquinone were dissolved in 0.6 ml warm AR ethanol, and added to the dialysed alkaline phosphatase.
  • J5 benzoquinone-activated alkaline phosphatase thus produced was sufficient for six 1.5 mg antibody conjugations.
  • Monoclonal antibody was dialysed against 2 x 500 ml of 0.15M sodium chloride at +4 C. Dialysed antibody was added to 4 mg of benzoquinone-activated alkaline
  • Example 35 antigen; the monoclonal of Example 6 was specific to IS. coli 06 (NCTC 11105, 9006); the monoclonal of Example 20 was specific to E. coli strains 9027 and E18535 both bearing the 027 antigen; the monoclonal of Example 29 was specific to E. coli strains 10964 and E32157/o both bearing the 0157 antigen; the monoclonal of Example 32 was specific to those strains of 33. coli bearing the CS2 antigen, C19f, 201 74 B34334f; the monoclonal of Example 33 was specific to 13.
  • Example 34 was specific to IS. coli strains NCTC 11100, 9006, 9022 and 10430 which all bear the HI antigen; the monoclonal of Example 38 showed a strong reaction against all sub-units of IS. coli K88; the monoclonal of Example 40 is specific to E_. coli labile endotoxin of human and porcine origin, and probably recognises the alpha-subunit of the protein; and the monoclonal of Example 41 is possibly specific to the beta-subunit of the E. coli LT of human origin.
  • Example 43 the monoclonals of Examples 8, 14, 18, 23, 29 and 38 were negative with respect to Serratia
  • the monoclonal of Example 8 was negative with respect to Campylobacter
  • the monoclonals of Examples 20 and 21 were negative with respect to Streptococcus
  • the monoclonals of Examples 30 and 41 were negative with respect to Citrobacter
  • the monoclonals of Examples 40, 41 and 42 were negative with respect to cholera toxin.
  • the Sub-class was IgM for Examples 2, 10, 16, 18, 21, 26, 27, 28 and 29, IgG3 for Examples 3, 4, 5, 9, 11, 12, 13, 14, 19, 20, 23, 30 and 36, IgG2a for Examples 6, 19, 24, 35 and 38, and IgGl for Examples 8, 17, 22, 32, 39, 40, 41 and 42.
  • the monoclonals of Examples 7 and 37 were positive with Protein A, indicating the S ⁇ b-class IgG2a, IgG2b or IgG3.
  • Example 1 The general procedure of Example 1 is used in preparing a monoclonal antibody broadly cross-reactive with antigens of the Escherichia genus.
  • Tests using the present invention are superior to 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 expenses; and (v) improved therapy based upon early, precise diagnosis.

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Abstract

Des anticorps monoclonaux pour le genre Escherichia, les anticorps marqués, compositions et kits les contenant, et leur utilisation pour le diagnostic d'antigènes et le traitement.
PCT/GB1985/000408 1984-09-07 1985-09-09 Anticorps monoclonaux et leur utilisation WO1986001806A1 (fr)

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GB8422653 1984-09-07
GB848422653A GB8422653D0 (en) 1984-09-07 1984-09-07 Monoclonal antibodies

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EP (1) EP0192726A1 (fr)
JP (1) JPS62500173A (fr)
GB (1) GB8422653D0 (fr)
WO (1) WO1986001806A1 (fr)

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EP0277697A1 (fr) * 1987-01-05 1988-08-10 University Of Pittsburgh Antigène de polymyxine lipopolysaccharide et méthode associée
EP0547137A1 (fr) * 1990-08-31 1993-06-23 Bristol-Myers Squibb Company Immunoglobulines homoconjuguees
WO1997018841A1 (fr) * 1994-07-01 1997-05-29 The Board Of Trustees Of The Leland Stanford Junior University Localisation non invasive d'un conjugue photoemetteur chez un mammifere
EP0969018A1 (fr) * 1998-07-02 2000-01-05 Combact Diagnostic Systems Ltd. Anticorps monoclonaux antibactèriens
US6217847B1 (en) 1994-07-01 2001-04-17 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6638752B2 (en) 1996-04-19 2003-10-28 Xenogen Corporation Biodetectors targeted to specific ligands
US7198774B2 (en) 1994-07-01 2007-04-03 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
KR20160117467A (ko) * 2014-02-06 2016-10-10 알사니스 바이오사이언시스 게엠베하 이. 콜라이 특이성 항체 서열
US10206992B2 (en) 2013-01-17 2019-02-19 Arsanis Biosciences Gmbh MDR E. coli specific antibody
US11844766B2 (en) 2016-10-24 2023-12-19 Janssen Pharmaceuticals, Inc. ExPEC glycoconjugate vaccine formulations
US11931405B2 (en) 2019-03-18 2024-03-19 Janssen Pharmaceuticals, Inc. Bioconjugates of E. coli O-antigen polysaccharides, methods of production thereof, and methods of use thereof

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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0277697A1 (fr) * 1987-01-05 1988-08-10 University Of Pittsburgh Antigène de polymyxine lipopolysaccharide et méthode associée
EP0547137A1 (fr) * 1990-08-31 1993-06-23 Bristol-Myers Squibb Company Immunoglobulines homoconjuguees
EP0547137A4 (en) * 1990-08-31 1993-12-08 Bristol-Myers Squibb Company Homoconjugated immunoglobulins
WO1997018841A1 (fr) * 1994-07-01 1997-05-29 The Board Of Trustees Of The Leland Stanford Junior University Localisation non invasive d'un conjugue photoemetteur chez un mammifere
US8545814B2 (en) 1994-07-01 2013-10-01 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6217847B1 (en) 1994-07-01 2001-04-17 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US7255851B2 (en) 1994-07-01 2007-08-14 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6649143B1 (en) 1994-07-01 2003-11-18 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6890515B2 (en) 1994-07-01 2005-05-10 Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6908605B2 (en) 1994-07-01 2005-06-21 Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6916462B2 (en) 1994-07-01 2005-07-12 Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6923951B2 (en) 1994-07-01 2005-08-02 Board Of Trustees Of The Leland Stanford University Non-invasive localization of a light-emitting conjugate in a mammal
US6939533B2 (en) 1994-07-01 2005-09-06 Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US7198774B2 (en) 1994-07-01 2007-04-03 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6638752B2 (en) 1996-04-19 2003-10-28 Xenogen Corporation Biodetectors targeted to specific ligands
US8349602B1 (en) 1996-04-19 2013-01-08 Xenogen Corporation Biodetectors targeted to specific ligands
EP0969018A1 (fr) * 1998-07-02 2000-01-05 Combact Diagnostic Systems Ltd. Anticorps monoclonaux antibactèriens
US10206992B2 (en) 2013-01-17 2019-02-19 Arsanis Biosciences Gmbh MDR E. coli specific antibody
US10940191B2 (en) 2013-01-17 2021-03-09 X4 Pharmaceuticals (Austria) GmbH MDR E. coli specific antibody
US11529405B2 (en) 2013-01-17 2022-12-20 Janssen Pharmaceuticals, Inc. MDR E. coli immunogen
KR20160117467A (ko) * 2014-02-06 2016-10-10 알사니스 바이오사이언시스 게엠베하 이. 콜라이 특이성 항체 서열
KR102301188B1 (ko) * 2014-02-06 2021-09-13 엑스4 파마슈티컬스 (오스트리아) 게엠베하 이. 콜라이 특이성 항체 서열
US11844766B2 (en) 2016-10-24 2023-12-19 Janssen Pharmaceuticals, Inc. ExPEC glycoconjugate vaccine formulations
US11931405B2 (en) 2019-03-18 2024-03-19 Janssen Pharmaceuticals, Inc. Bioconjugates of E. coli O-antigen polysaccharides, methods of production thereof, and methods of use thereof

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EP0192726A1 (fr) 1986-09-03
GB8422653D0 (en) 1984-10-10
JPS62500173A (ja) 1987-01-22

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