Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to monoclonal antibodies and their use. BACKGROUND OF THE INVENTION
• Organisms (gram-negative) of the genus Mobiluncus are inhabitants of the vaginal tract. They are implicated in vaginosis syndrome.
• Species of the genus are Mobiluncus mulieris and Mobiluncus curtisii. Among the latter, there are subspecies holmesii and curtisii.
• the present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Mobiluncus antigens and/or organisms.
• the present invention comprises monoclonal antibodies specific for an antigen of Mobiluncus; in particular, the antigens of Mobiluncus mulieris and Mobiluncus curtisii (inc. subsp. holmesii and curtisii) , as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Mobiluncus.
• the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Mobiluncus antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens or to a particular species thereof and having linked thereto an appropriate label.
• the label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom, or particle, or any other label.
• the invention further comprises the process for diagnosing the presence of Mobiluncus antigens or organisms in a specimen comprising contacting said specimen with the labelled monoclonal antibody in an appropriate im unoassay procedure.
• the invention is also directed to a therapeutic composition
• a therapeutic composition comprising a monoclonal antibody for an antigen of Mobiluncus and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of Mobiluncus.
• the monoclonal antibodies of the present invention • are prepared by fusing spleen cells, from a mammal which has been immunised against the particular Mobiluncus antigen, with an appropriate myeloma cell line, preferably NSO (uncloned) , P3NS1-Ag4/1, or Sp2/0 Agl4.
• 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.
• HAT hypoxanthine, aminopterin and thymidine
• the immunised spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice. rats, 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 chosen Mobiluncus antigen over a suitable period, e.g. 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 Mobiluncus 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 give a positive response to the presence of the particular Mobiluncus 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 Mobiluncus antigen.
• the monoclonal antibody selected, which is specific for the particular Mobiluncus 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 a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
• labels such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
• the present invention will be described with reference to the use of an enzyme labelled monoclonal antibody.
• Some of the enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, urease, and the like.
• Such linkage with enzymes can be accomplished by any one of the conventional and known methods, 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 non-labelled 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 heterogenous 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 nano-mole 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 Mobiluncus 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 Mobiluncus infections and they are used in amounts effective to cure; an amount which will vary widely dependent 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 Mobiluncus in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Mobiluncus alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Mobiluncus and/or other bacteria. In the past there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens such as 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 can 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 Mobiluncus 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 Mobiluncus 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.
• kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Mobiluncus.
• kits comprising at least one labelled monoclonal antibody against a particular Mobiluncus antigen or species, as well as any appropriate stains, counterstains, or reagents.
• Specific antigens to be detected in this kit include the antigen of Mobiluncus mulieris or Mobiluncus curtisii (or a subspecies thereof) .
• Monoclonal diagnostics which detect the presence of Mobiluncus 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 Mobiluncus antigen. More particularly, products of 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 contamina ⁇ tion.
• 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
• Mobiluncus curtisii subsp. curtisii antigen was obtained from the National Collection of Type Cultures (NCTC 11656) . It may be tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . More specifically, the antigen may be removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity. The blood agar is scraped off into formol saline, as a source of antigen. The organisms were washed in formol saline by repeated centrifugation and were finally resuspended in formol saline.
• mice Animal Immunisation Balb/c mice were injected with the prepared antigen. They were given two im injections + CFA, with an interval of 2 weeks between the intramuscular injections. The mice were rested for 6 weeks before being given a further iv injection + PBS. 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. 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.
• myeloma cell line NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England, myeloma cells were obtained 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 was 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 wells were kept undisturbed and cultured at 37°C in 9% CO « air at approximately 100% humidity.
• the wells were analysed for growth utilising the conventional inverted microscope procedure, after about 5 to 10 days.
• Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtiter plate. The plates were incubated for 7-14 days at 37°C, 95% RH, 7-9%
• the monoclonal antibodies from the clones were screened by the standard techniques for binding to the Mobiluncus antigen prepared as in the immunisation, and for specificity in a test battery of Mobiluncus species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms was prepared, boiled, and utilised as a template to define the specificity of the parent O-specific group. The EIA immunoassay noted above was used.
• the monoclonal antibody specific against the Mobiluncus antigen prepared and screened as described above, was then bound to an appropriate enzyme (in this case, a highly purified alkaline phosphatase) .
• an appropriate enzyme in this case, a highly purified alkaline phosphatase
• alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 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. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. Unreacted benzoquinone and reaction by-products were then 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 4 mg of benzoquinone-activated alkaline phosphatase and immediately followed by sufficient IM sodium bicarbonate to give a final concentration of 0.1M.
• the conjugation mixture was left in the dark at +4 C for 48 hours. Sufficient IM lysine was then added to give a final concentration of 0.1M. After 2 hours in the dark at room temperature, the conjugate was dialysed against 2 x 1000 ml 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 of Mobiluncus.
• the monoclonal antibody was also tested and shown to be of the immunoglobulin Class.
• 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.
• the antibody can first be bound to the plate.
• Example 2
• Example 2 The same procedure as in Example 1 may be utilised to prepare a monoclonal antibody from M. curtisii subsp. holmes (NCTC 11657) , with the exceptions that the second im injection used IFA rather than CFA, the rest period p between im and iv injections was 7 weeks, 2.5 x 10
• Example 3 240 wells were used. The subclass is IgGl. The same cross-reactivity is observed as in Example 1.
• Example 3 The same cross-reactivity is observed as in Example 1.
• Example 2 The same procedure as in Example 2 may be utilised to prepare a monoclonal antibody from M. mulieris (NCTC 11658) , with the exceptions that the myeloma cells were
• Antibodies of subclasses IgGl and IgG2a were obtained, specific to the NCTC 11658 antigen.
• Example 4 Antibodies of subclasses IgGl and IgG2a were obtained, specific to the NCTC 11658 antigen.
• Example 1, 2 or 3 may be utilised to prepare a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Mobiluncus, but using another Mobiluncus antigen 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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• 1986
  • 1986-04-25 GB GB868610204A patent/GB8610204D0/en active Pending
• 1987
  • 1987-04-27 WO PCT/GB1987/000278 patent/WO1987006468A1/en unknown
  • 1987-04-27 EP EP19870902630 patent/EP0265478A1/de not_active Withdrawn

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