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
  • 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.
• 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.
• 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 Providencia antigens and/or organisms.
• the present invention comprises monoclonal antibodies specific for an antigen of Providencia; in particular, the antigens of Providencia stuartii, the antigens of Providencia rettgeri, and the antigens of Providencia alcalifaciens, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Providencia.
• the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Providencia antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Providencia 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 Providencia 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 Providencia and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Providencia.
• the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Providencia 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 Providencia 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 Providencia 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 Providencia 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 Providencia antigen.
• the monoclonal antibody selected, which is specific for the particular Providencia antigen or species, is then bound to an appropriate label.
• Amounts of antibody sufficient fox 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.
• One example is the enzyme-linked immunosorbent. ssay (EIA) .
• 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 im unoglobin 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. In both chemiluminescent and bioluminescent ' reactions, 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 Providencia 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 Providencia 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 Providencia in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Providencia alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Providencia and/or other bacteria.
• 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 Providencia 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 Providencia 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 Providencia.
• One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Providencia antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing a ' t least one control sample of a Providencia antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Providencia organisms in a particular sample.
• Monoclonal diagnostics which detect the presence of Providencia 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 Providencia 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 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
• Providencia stuartii was obtained from the National Collection of Type Cultures (NCTC accession No. 10318) and tested against standard reference typing sera to confirm its typing. More specifically, the antigen 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 formol saline by repeated centrifugation and were resuspended in formol saline.
• mice Six Balb/c mice were injected with the prepared antigen. They were given one intraperitoneal injection per week for three weeks (0.05 ml 80% T vaccine) followed by five intravenous injections every week of 80% T boiled killed Providencia stuartii prepared as above, for a further three 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. 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 (0.05 ml of 80% T vaccine) intravenously, three days prior to splenectomy.
• 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 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
• 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.
• 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.
• 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 the antigen prepared as in the immunisation, and for specificity in a test battery of Providencia species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms, i.e. Providencia, Pseudomonas, Proteus, E. coli, Serratia and Enterobacter, 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 fluid was titrated, as noted above, to establish presence and level of antibody, and purified. Purification is 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 solution was dialysed vs 2 x 1000 ml of the same buffer at +4°C.
• the dialysed, redissolved precipitate was centrifuged at 30,000 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.
• 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 ⁇ l 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 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 and 3
• Example 1 The procedure of Example 1 was followed in each case, with differences outlined below, to prepare monoclonal antibody conjugates for various antigens of the genus Providencia.
• the mice were rested for a month after the intravenous injection regimen and then revaccinated with 0.05 ml 80% T vaccine.
• Example 3 In the animal immunisation step for Example 3, the intraperitoneal injection per week for 3 weeks was followed by an intravenous injection per week for 4 weeks, of boiled, kiled cells.
• Example 2 Providencia alcalifaciens NCTC 10286 was used; in Example 3, Providencia rettgeri NCTC 7475.
• the limiting dilution method was used; in this method, 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.
• Example 2 Enterobacter and Providencia were used in Example 2 and Proteus in Example 3.
• the benzoquinone method was used. 24 mg alkaline phosphatase (Sigma Type VII-T) were dialy ⁇ ed 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 benzoguinone/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
• 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 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 Providencia, but using another 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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