WO1986000644A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation Download PDF

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Publication number
WO1986000644A1
WO1986000644A1 PCT/GB1985/000294 GB8500294W WO8600644A1 WO 1986000644 A1 WO1986000644 A1 WO 1986000644A1 GB 8500294 W GB8500294 W GB 8500294W WO 8600644 A1 WO8600644 A1 WO 8600644A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
serratia
antigen
immunoassay
enzyme
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PCT/GB1985/000294
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English (en)
Inventor
Bruce William Wright
Peter John Cox
Alice Margaret Noyes
Danny Widdows
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Technology Licence Company Limited
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Publication date
Application filed by Technology Licence Company Limited filed Critical Technology Licence Company Limited
Publication of WO1986000644A1 publication Critical patent/WO1986000644A1/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.
  • Serratia is described in Zinsser Microbiology (17th ed.) 730-1.
  • the organisms of the genus Serratia were at one time thought to be harmless saphrophytes and were used to determine air current patterns in hospitals. These organisms, however, are rapidly emerging as major entities in nosocomial infections. Almost all Serratia infections are associated with underlying disease, changing physiological patterns, immunosuppressive treatment, or mechanical manipulations of the patient. In nature, Serratia are found in soil, water, plants and animals. The are frequently isolated from insects and may play a role in diseases of certain insects.
  • Serratia Divisions have been made among the Serratia species.
  • the more commonly known Serratia members include Serratia marcescens, Serratia liquefaciens and Serratia rubidaea.
  • the extreme specificity of antigen-antibody reactions has made it possible to recognise differences between strains of a bacterial species, such as the above-mentioned species, that are indistinguishable on the basis of other phenotypic criteria.
  • S_. marcescens is the most frequent isolate of the genus Serratia and has been associated with a number of nosocomial epidemics involving pneumonia, septicemia, urinary tract infections and wound infections.
  • Serratia 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. At the present time, the test for gram-negative sepsis involves processing blood and urine cultures and other procedures on occasion. In addition to being expensive, 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.
  • 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 Serratia antigens and/or organisms.
  • the present invention comprises monoclonal antibodies specific for an antigen of Serratia; in particular, the antigens of Serratia marcescens, the antigens of Serratia liquefaciens, and the antigens of Serratia rubidaea, • as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Serratia.
  • the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Serratia antigens, each comprising- a monoclonal antibody against one of the above-mentioned antigens to Serratia 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 Serratia antigens or organisms in a specimen, comprising contacting said specimen with the labelled monoclonal antibody in an appropriate i munoassay procedure.
  • the invention is also directed to a therapeutic composition
  • a therapeutic composition comprising a monoclonal antibody for an antigen of Serratia and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Serratia.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Serratia 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 Serratia 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 Serratia 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 Serratia 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 Serratia antigen.
  • the monoclonal antibody selected, which is specific for the particular Serratia 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.
  • 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
  • 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.
  • 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 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 lu inol.
  • 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 Serratia 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 Serratia 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 Serratia in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Serratia alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Serratia 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. For example, a kit 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 Serratia 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 Serratia 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 Serratia.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Serratia antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Serratia antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Serratia organisms in a particular sample.
  • Monoclonal diagnostics which detect the presence of Serratia 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 Serratia antigen.
  • 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 monoclonal antibodies of the present invention were prepared generally according to the method of Kohler and Milstein, supra. In the Examples:
  • 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
  • API biochemical
  • mice Six Balb/ ⁇ mice were- injected with the prepared antigen. They were given 3 weekly intraperitoneal injections (0.05 ml 80% T vaccine) of boiled killed Serratia marcescens prepared as above, for a total of six 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 i munosorbent assay system. 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 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 ⁇ re 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 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.
  • each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin, and thymidine) and a feeder layer of Balb/c
  • FCS-DMEM was spread evenly over the base. The cells were allowed to multiply for approximately 10 days at 37°C, 7-9% C0 2 , 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.
  • dilutions of cell suspensions in 18% FC-DMEM + Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtitre plate.
  • the plates were incubated for 7-14 days at 37°C, 95% RH, 7-9% C0 2 , until semi-confluent.
  • the supernatants were then 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 the ' antigen prepared as in the immunisation, and for specificity in a test battery of Serratia species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms, i.e. Proteus and Enterobacter, was prepared, boiled, and utilised as a template to define the specificity of the parent
  • the fluid was titrated, as noted above, to establish presence and level of antibody, and purified. Purification is accomplished using the supernatant on Protein A-Sepharose method, in which, 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/ in 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 1M Tris buffer, pH 9.0, to raise the pH immediately to about 7.5.
  • citrate buffer 0.1M sodium citrate, pH 3.5
  • 1M Tris buffer pH 9.0
  • the eluate was dialysed in PBS, pH 7.4, at 4°C, and stored at -20°C.
  • the monoclonal antibody specific against Serratia marcescens antigen prepared and screened as described above, is .then bound to an appropriate enzyme; in this case, a highly purified alkaline phosphatase. This is accomplished b the benzoquinone conjugation method.
  • 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. After this time, unreacted • benzoquinone and reaction by-products were removed and the buffer exchanged, by gel filtration on a Pharmacia
  • PD-10 (Sephadex G-25M) column previously equilibrated in 0.15M sodium chloride.
  • the 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 8 mg of benzoquinone-activated alkaline phosphatase, immediately followed by sufficient 1M sodium bicarbonate to give a final concentration of 0.1M..
  • the conjugation mixture was left in the dark at +4°C for 48 hours. After this time, sufficient 1M lysine was added to give a final concentration of 0.1M.
  • 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.
  • 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 antigen Serratia marcescens.
  • 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.
  • Example 2 Following the procedure of Example 1, conducting animal immunisation differently, without using the dilution method in cloning, conducting antibody production and antibody purification differently, a monoclonal antibody of the class IgG2a was obtained.
  • the tested organisms were Enterobacter and Proteus; there was reaction with two strains of Serratia marcescens, one strain of Serratia marinorubra and Erwinia herbicola (Enterobacter agglomerans) .
  • mice were vaccinated intraperitoneally with the prepared antigen (0.05 ml of 80% T vaccine) followed by an intravenous boost after six weeks. When the mice showed a suitably elevated titre to the immunising antigen, they were used as a source of spleen cells for fusion.
  • Balb/c mice were primed with pristane, for at least 7 days, and were injected with 10 cells of the monoclonal antibody producing cell line. Ascitic fluid was harvested when the mice were swollen with fluid but still alive. The fluid was centrifuged at 1200 g for approximately 10 min, the cells discarded and the antibody-rich ascites collected and stored at -20°C. In antibody purification, the Protein A-Sepharose method was used. Ascites fluid was filtered through glass wool and centrifuged at 30,000 f for 10 min. The ascites was then diluted with twice its own volume of cold phosphate buffer. (CIM. sodium phosphate, pH .8.2) .
  • CCM cold phosphate buffer
  • the diluted ascites was applied to a 2 ml column of Protein A-Sepharose, previously equilibrated with phsophate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient 1M Tris buffer, pH 9.0 to raise the pH immediately to about 7.5. The eluate was dialysed in phosphate buffered saline, pH 7.4 at 4°C and stored at
  • Example 3 The same procedure as in Example 1 is utilized, in preparing .the monoclonal antibodies specific for the antigens of Serratia liquefaciens, NCTC 10861, and Serratia marinorubra, NCTC 10912.
  • intraperitoneal injections were given weekly for 3 weeks followed by an intravenous injection per week for 3 weeks, then one intravenous injection a month later and one intravenous injection 4 months later.
  • intraperitoneal injections were given weekly for 3 weeks followed by an intravenous injection per week for 3 weeks, then one intravenous injection a month later and one intravenous injection 4 months later.
  • Example 7 5 x 10 myeloma cells were used.
  • the intraperitoneal injections were given each week for 6 weeks and were followed by a further intravenous injection a month later.
  • Example 5
  • Example 2 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. Serratia, but using another Serratia 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 labor required to administer laboratory procedures, resulting in reduced labor- 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. While the invention has been described in connection with certain preferred embodiments, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

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  • Chemical & Material Sciences (AREA)
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Abstract

Anticorps monoclonaux du genre Serratia, anticorps étiquetés, compositions et kits les contenant, et leur utilisation dans le diagnostic d'antigènes et dans le traitement.
PCT/GB1985/000294 1984-07-03 1985-07-02 Anticorps monoclonaux et leur utilisation WO1986000644A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB848416843A GB8416843D0 (en) 1984-07-03 1984-07-03 Monoclonal antibodies
GB8416843 1984-07-03

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WO1986000644A1 true WO1986000644A1 (fr) 1986-01-30

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JP (1) JPS61502631A (fr)
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WO (1) WO1986000644A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0077734A2 (fr) * 1981-10-19 1983-04-27 Mgi Pharma, Inc. Production d'anticorps monoclonaux contre des adhésines bactériennes
WO1983001739A1 (fr) * 1981-11-17 1983-05-26 Brigham & Womens Hospital Anticorps monoclonaux contre le brugia malayi
EP0101039A2 (fr) * 1982-08-10 1984-02-22 Meiji Seika Kabushiki Kaisha Anticorps monoclonal, méthode pour sa production et son utilisation
EP0105714A1 (fr) * 1982-09-29 1984-04-18 Serono Diagnostics Limited Immunoessai pour antigènes
EP0111762A1 (fr) * 1980-06-20 1984-06-27 Unilever Plc Procédés et appareil pour l'exécution d'essais de liaisons spécifiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0111762A1 (fr) * 1980-06-20 1984-06-27 Unilever Plc Procédés et appareil pour l'exécution d'essais de liaisons spécifiques
EP0077734A2 (fr) * 1981-10-19 1983-04-27 Mgi Pharma, Inc. Production d'anticorps monoclonaux contre des adhésines bactériennes
WO1983001739A1 (fr) * 1981-11-17 1983-05-26 Brigham & Womens Hospital Anticorps monoclonaux contre le brugia malayi
EP0101039A2 (fr) * 1982-08-10 1984-02-22 Meiji Seika Kabushiki Kaisha Anticorps monoclonal, méthode pour sa production et son utilisation
EP0105714A1 (fr) * 1982-09-29 1984-04-18 Serono Diagnostics Limited Immunoessai pour antigènes

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EP0189450A1 (fr) 1986-08-06
JPS61502631A (ja) 1986-11-13

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