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
  • C07K16/1235—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 Salmonella (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.
• Salmonella species are typed according to group antigens.
• Salmonellosis probably represents the largest single communicable bacterial disease problem in the United States. Contaminated food and water are the mechanisms of transmission for all Salmonella, including £. typhi.
• typhoid fever In ⁇ .• typhi infection (typhoid fever) , the human carrier is the source; man is the only known host. In other salmonelloses, animals are more important. Food-borne salmonellosis is quite common and, for the non-typhoidal types, probably constitutes the major source of infection today. . typhi also can spread via food or water.
• the actual disease process may be apparent as any of three distinct clinical entities: a gastroenteritis, a septicemia with focal lesions, or an enteric fever such as typhoid fever.
• Salmonella gastroenteritis like shigellosis, represents an actual infection of the bowel and usually occurs about 16 hours after ingestion of the organism. The disease is characterised by diarrhea, fever and abdominal pain which is usually self-limiting and lasts for two to five days.
• Salmonella septicemia is prolonged and characterised by fever, chills, anorexia and anaemia. Focal lesions may develop in any tissue, producing osteomyelitis, pneumonia, pulmonary abscesses, meningitis or endocarditis. Gastroenteritis is minor or even absent, and the organism is rarely isolated from the faeces.
• S_. cholerae-suis is a frequent isolate from this type of disease. Osteomyelitis in persons with sickle cell trait is most frequently caused by S_. cholerae-suis.
• the prototype and most severe enteric fever is typhoid fever, and the causative agent is S_. typhi.
• Other Salmonella particularly serotypes paratyphi A and paratyphi B, also can cause enteric fevers, but the symptoms are milder and the mortality rate is lower.
• Various vaccines have been tried to control typhoid fever. While some encouraging results have been obtained by use of the K vaccine (containing Vi antigen) in children living in endemic areas, no vaccine has proved to be entirely successful in preventing disease, especially when the number of ingested organisms is large. A more practical approach to prevent spread of typhoid fever is to insure safe drinking water supplies.
• Salmonella is known to cause bacterial diarrhea. Bacterial diarrhea is a common and often serious condition manifest as fluid loss from the bowel, leading in many cases to dehydration, and occasionally death.
• Salmonella In addition to diarrhea. Salmonella 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.
• 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.
• the present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Salmonella antigens and/or organisms.
• the present invention comprises monoclonal antibodies specific for an antigen of
• Salmonella in particular, the somatic antigens of groups A to Z and 51 to 65, the H (flagellar) antigens A to Z and Zl to Z59; the antigens of Salmonella albany. Salmonella poona and Salmonella brazil; the invasiveness antigens 1 and 2, attachment antigens 1 and 2, and enterotoxins 1 and 2; as well as a monoclonal antibody broadly cross-reactive with an antigen for each species (or substantially-all species) of the genus Salmonella.
• the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Salmonella antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Salmonella 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 Salmonella 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 Salmonella and a carrier or diluent, as -b-
• kits containing at least one labelled monoclonal antibody to an antigen of a Salmonella well as kits containing at least one labelled monoclonal antibody to an antigen of a Salmonella.
• the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Salmonella 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.
• 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 Salmonella antigen chosen, e.g. 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 Salmonella 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 Salmonella 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 Salmonella antigen.
• the monoclonal antibody selected, which is specific for the particular Salmonella antigen or species, is then bound to an appropriate label. Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture _in vivo in mammals such as mice.
• the monoclonal antibodies may be labelled with various labels, as exemplified above. The present invention will be described with reference to the use of an enzyme-labelled monoclonal antibody. Examples of enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase and urease.
• Such linkage with enzymes can be accomplished by any known method, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
• testing is carried out employing one of a wide variety of conventional immunoassay methods. The particular method chosen will vary according to the monoclonal antibody and the label chosen.
• enzyme immunoassays are preferred owing to their low cost, reagent stability, safety, sensitivity and ease of procedure.
• EIA enzyme-linked immunosorbent assay
• EIA is a solid-phase assay system which is similar in design to the radiometric assay, but which utilises an enzyme in place of a radioactive isotope as the immunoglobin marker.
• Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluorescent probes. A non-labelled antigen and a specific antibody are combined with identical fluorescently-labelled antigen. Both labelled and unlabelled antigen compete for antibody binding sites. The amount of labelled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of non-labelled antigen. Examples of this particular type of fluorescent-immunoassay include heterogeneous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate-Labelled Fluorescent Immunoassay. The most suitable fluorescent probe, and the one most widely used, is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimised for the probe utilised in the particular assay, and in which the effect of scattering can be minimised.
• Fluorescence polarisation In fluorescence polarisation, a labelled sample is excited with polarised light and the degree of polarisation of the emitted light is measured. As the antigen binds to the antibody, its rotation slows down and the degree of polarisation increases. Fluorescence polarisation is simple, quick and precise. However, at the present time, its sensitivity is limited to the micro ole 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.
• 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.
• a catalytic protein or enzyme such as luciferase
• 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 Salmonella ⁇ _-,_ . _, PCT/GB85/00407 86/01805 '
• compositions can be used to treat humans and/or animals afflicted with some form of shigellosis and they are used in amounts effective to cure; the amount may vary widely, depending upon the individual being treated and the severity of the infection.
• One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens or species of
• Salmonella in various specimens It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Salmonella alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Salmonella and/or other bacteria.
• conjugated or labelled monoclonal antibodies for antigens and/or species of Salmonella 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 Salmonella 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 Salmonella.
• One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Salmonella antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Salmonella antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Salmonella organisms in a particular sample.
• Monoclonal diagnostics which detect the presence of Salmonella 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 Salmonella 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.
• 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
• Salmonella paratyphi A was obtained from the National Collection of Type Cultures (NCTC accession No. 13) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) .
• the Salmonella 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 for ol saline, and then resuspended in 1% formol saline.
• mice Six Balb/c mice were injected with the prepared antigen, over five weeks. They were given one ip injection per week for three weeks (0.05 ml 80% T vaccine) of boiled killed Salmonella OAl, followed by a 0.05 ml dose of vaccine iv each week for two 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 (0.02 ml 80% T vaccine) intravenously, three days prior to splenectomy.
• Spleen cells from the immune mice were harvested three days after boosting, by conventional techniques. First, 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 with Methyl Violet-citric acid solution, and a haemacytometer and light microscope were used to count the stained nuclei of the cells.
• 10 ml serum-free tissue culture medium DMEM were then slowly added, followed by up to 50 ml of such culture medium, centrif gation and removal .of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight FCS.
• 10 ⁇ l of the mixture were placed in each of 480 wells of standard multiwell tissue culture plates. Each well contains 1.0 ml of the standard HAT medium
• 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. in those wells in which growth was present in the inhibiting HAT medium, screening tests for the specific monoclonal antibody were made utilising the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the Salmonella paratyphi A antigens was developed in at least one well. D. Cloning
• 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 Salmonella OAl NCTC 13, prepared as in the immunisation, and for specificity in a test battery of Salmonella -44-
• the monoclonals had the appropriate specificity (to S_. paratyphi A) , and did not react with Shiqella, 12. coli, Pseudomonas, Klebsiella, Serratia or Enterobacter. F. Antibody production and purification
• DMEM monoclonal antibody-producing line specific to the Salmonella paratyphi A antigen were grown in batch tissue culture.
• DMEM to which had been added 10% FCS, was used to support growth in mid-log phase, to 1 litre volume. The culture was allowed to overgrow, to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes. The cell/cell debris was discarded and the antibody-rich supernatant collected. The fluid may then be titrated, as noted above, to establish presence and level of antibody, and purified by a combination of batch ion-exchange chromatography, ammonium sulphate precipitation and column ion-exchange chromatography.
• 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.
• 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 monoclonal antibody specific against Salmonella paratyphi A was linked to an enzyme, viz. highly-purified alkaline phosphatase, using the one-step glutaraldehyde method.
• 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 -46-
• 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
• Monoclonal antibody was produced and found to be specific for the group A antigens of Salmonella. The monoclonal antibody was tested and shown to be of the Class IgG_.
• 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 7
• Example 2 The procedure of Example 1 was followed in each of 6 cases, with differences outlined below, to prepare monoclonal antibodies and conjugates for various antigens of the genus Salmonella.
• the antigen was Salmonella hadar, NCTC 9877; in Example 3, Salmonella albany (formerly K3) NCTC 9869; in Example 4, Salmonella brazil NCTC 8446; in Example 5, Salmonella poona, NCTC 4840; and, in Examples 6 and 7, Salmonella enteriditis, NCTC 5188.
• DMEM was the growth medium, and the organisms were merely washed once in formol saline.
• Example 8 In the cell fusion step, 1 x 10 myeloma cells, were used in Example 2, and 6 x 10 in Examples 3, 5 and 6. t " 1.2 x 10 8 spleen cells were used in Example 2, 1.4 x 108 in Examples 3 and 5.
• Example 5 Additionally in Example 2, and as the antibody production procedure in Examples 3, 4, 5 and 7, Balb/c mice were primed with pristane. for at least 7 days, and
• Antibody purification for Examples 3 and 4 was by 5 the ammonium sulphate precipitation/DEAE-cellulose 86/01805 ' '
• 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.l M 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 phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1 M sodium citrate, pH 3.5) into sufficient 1 M TRIS buffer, pH 9.0, to raise the pH immediately to about 7.5. The eluate was dialysed in PBS, pH 7.4, at 4 C, and stored at -20 C.
• citrate buffer 0.1 M sodium citrate, pH 3.5
• the benzoquinone method was used. 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25M sodium phosphate buffer, pH 6.0, at +4°C. 18 ' mg p-benzoquinone, 18 mg were dissolved in 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.
• 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 2 was specific to Salmonella newport and Salmonella hadar.
• the monoclonal of Example 6 is specific to Salmonella enteriditis which bears the 9,12 antigens representative of Salmonella OD1.
• the monoclonal antibodies of Examples 2, 3, 4 and 5 are negative to other Salmonella, 33. coli and Shigella, and the monoclonals of Examples 2, 4 and 5 are also negative with respect to Serratia, Enterobacter, Pseudomonas and Klebsiella.
• the monoclonal of Example 7 cross-reacts with all Salmonella strains tested to date.
• the monoclonal antibodies obtained in Examples 2 to 7 are respectively of the Sub-classes IgGl, IgM, IgG2a and IgG3, IgG3, IgM and IgG2a.
• 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. 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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• 1984
  • 1984-09-07 GB GB848422650A patent/GB8422650D0/en active Pending
• 1985
  • 1985-09-09 EP EP19850904473 patent/EP0193576A1/de not_active Withdrawn
  • 1985-09-09 WO PCT/GB1985/000407 patent/WO1986001805A1/en not_active Application Discontinuation
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