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
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (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 specific for the antigens or species of Strepto ⁇ cocci are desired which when used will rapidly diagnose the presence of such organisms in speci ⁇ mens.
• Strepto ⁇ coccus species Divisions have been made among the Strepto ⁇ coccus species.
• Some of the representative members include Streptococcus pneumoniae, j3. pyogenes, S_. aqalactiae, S_. bovis, S_. cremoris, j3. dysgalactiae, S_. fecalis, S_. faecium, S_. lactis, S_. equisimilis, S_. zooepidermicus, S_. equi, and S_. uberis.
• V homogeneity of the immunological groups with 5 the natural habitat of the bacteria.
• S_. pneumoniae While involved with very different disease processes from the beta hemolytic Streptococci, S_. pneumoniae is nevertheless included within the broad grouping of Streptococcal organisms.
• the erythrogenic toxin is a substance, which upon intradermal injection in man, gives rise to a marked local erythema. This toxin is antigenic, and relatively heat- resistant. Further, at least two types of filter-
• 25 able hemolysins are formed by Streptococci; Streptolysin S, which is sensitive to heat and acid, and Streptolysin 0, which is sensitive to oxygen, but resistant to heat and acid.
• Streptococ ⁇ ci will be described with particular reference to beta Streptococcus Group A, as it is one of the most deadly and lethal microorganisms to man. It is the common cause of skin infec- tions, producing a rapidly spreading cellulitus that may enter the blood stream and produce blood poisoning. Other diseases commonly caused by the beta Streptococcus Group A include strep throat, tonsillitis, streptococcal pneumonia, and all wound infections where severe results may ensue without prompt treatment. The diagnosis of Streptococcal infection in tissues may be sometimes difficult because the organism may not be identified.
• rheumatic fever is a regular accompaniment of Streptococcal infections. It appears to be an immune reaction to the organ- ism. It occurs days or weeks after the infection.
• the most common cause of valvular heart disease . . are several dozen types of beta Streptococcus Group A distinguished by different surface prop ⁇ erties; i.e., carbohydrates of Lancefield's groups, proteins, T antigens, and R antigens. A small number of these may produce a severe kidney disease called glomerulonephritis. A somewhat larger number have been associated with rheumatic fever.
• isotopic and nonisotopic immunoassays have been utilized in conjunction with monoclonal antibodies to test for the pres ⁇ ence of an antigenic substance.
• agglutination, immuno-fluorescent, chemilum- inescent or fluorescent immunoassay, immuno- electron microscopy, radiometric assay systems, radio immunoassays, and enzyme-linked immunoassays are the most common techniques used with the monoclonal antibodies. Other techniques include bioluminescent, fluorescence polarization, and photon-counting immunoassays.
• EIA enzyme-linked immunoassay procedure
• the enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- bent assay procedure to determine the presence of an antigenic substance.
• the serotype of the infecting organism can be determined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.
• the present invention provides novel mono ⁇ clonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Streptococci antigens and/or organisms.
• the present invention com ⁇ prises monoclonal antibodies specific for an antigen or species of Streptococci; in particular, the antigens or species of Streptococcus pneumon ⁇ iae, S_. pyogenes, S_. agalactiae, S_. bovis, S_. cremoris, S_. dysgalactiae, S_. fecalis, S_. faecium, S_. lactis, S_. equisimilis, S_. zooepider- micus, j3.
• equi S ⁇ , uberis
• the antigens or species of beta Streptococcus specifically Groups A through G
• the antigen or antigens to the toxins of Streptococci such as the erythrogenic toxin and the streptolysins 0 and S
• a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Strepto ⁇ coccus.
• the invention also comprises labeled mono- clonal antibodies for use in diagnosing the presence of the Streptococci antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Streptococci or to a particular species thereof and linked thereto an appropriate label.
• the label can be chosen from the group consisting of a radio ⁇ active isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent com ⁇ pound, ferromagnetic atom, or particle, or any other label.
• the invention further comprises the process for diagnosing the presence of Streptococci antigens or organisms in a specimen comprising contacting said specimen with the labeled mono- clonal antibody in an appropriate immunoassay procedure.
• the invention is also directed to a therapeutic composition
• a therapeutic composition comprising a mono ⁇ clonal antibody for an antigen of Streptococcus and a carrier or diluent, as well as kits contain ⁇ ing at least one labeled monoclonal antibody .
• the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Streptococcus 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. Screening tests for the specific mono ⁇ clonal antibodies are employed utilizing immuno ⁇ assay techniques which will be described below.
• the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice.
• the mouse is first immunized by injection of the particular Streptococcus antigen chosen gener ⁇ ally for a period of approximately eleven weeks. When the mouse shows sufficient antibody produc ⁇ tion against the antigen, as determined by conven ⁇ tional assay, it is given a booster injection of the appropriate Streptococcus antigen, and then killed so that the immunized spleen may be removed. The fusion can then be carried out utilizing immunized 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 Streptococcus antigen are removed and cloned utilizing any of the standard methods.
• the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Streptococcus antigen.
• the monoclonal antibody selected, which is specific for the particular Streptococcus antigen or species, is then bound to an appropri ⁇ ate label. Amounts of antibody sufficient for labeling 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 labeled 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 labeled monoclonal antibody.
• Some of the enzymes utilized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, or 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
• Fluorescent-immunoassay is based on the labeling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labeled antigen. Both labeled and unlabeled antigen compete for antibody binding sites. The amount of labeled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen. Examples of this particular type of fluorescent- i munoassay would include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate Labeled Fluorescent Immunoassay. The most suit ⁇ able fluorescent probe, and the one most widely used is fluorescein. While fluore ' scein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimized for the probe utilized in the particular assay and in which the effect of scattering can be minimized.
• Fluorescence polarization In fluorescence polarization, a labeled sample is excited with polarized light and the degree of polarization of the emitted light is measured. As the antigen binds to the antibody its rotation slows down and the degree of polari ⁇ zation increases. Fluorescence polarization is simple, quick, and precise. However, at the present time its sensitivity is limited to the micromole per liter range and upper nano- mole per liter 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 inter ⁇ mediate 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 Streptococcus 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 Streptococci infections and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individ ⁇ ual being treated and the severity of the infec ⁇ tion.
• 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 Streptococcus in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Streptococcus alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Streptococci and/or other bacteria.
• kits In the past there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens with antiserum.
• the use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis.
• a rapid and precise kit could replace or augment existing tests and permit early direct therapy using precise antibiotics. Avoiding multiple antibiotics or more expensive or hazardous antibiotics would represent substantial patient and hospital sav- ings.
• a kit can be used on an out-patient basis. At present the lack of a rapid test giving "same day" answers may delay the initiation of treatment until the patient has developed more severe symptoms or may require the initiation of more costly therapy in a sick patient. A test that would return results within an hour or two would be a substantial convenience to patients.
• 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 Streptococci.
• kits comprising at least one labeled monoclonal antibody against a particular Streptococcus antigen or species, as well as any appropriate stains, counterstains, or reagents.
• Specific antigens to be detected in this kit include the common anti- gens of Streptococci, the antigens of S_. pneum ⁇ oniae, S_. pyogenes, S_. agalactiae, S_. bovis. S_.
• cremoris S_. dysgalactiae, S_. fecalis, S_. faecium, S_. lactis, S_. equisimilis, S_. zooepi- dermicus, S_. equi, S_. uberis, the antigens of beta Streptococcus, as well as the antigens to the toxins of Streptococci.
• Monoclonal diagnostics which detect the presence of Streptococci 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 labeled 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, milk, and the like contain the particular Strepto ⁇ coccus antigen. More particularly, the invention could be utilized as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamina ⁇ tion.
• 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
• Streptococcus antigen was obtained from the National Collection of Type Cultures and tested by standard biochemical methods of micr ⁇ bial identification to confirm its identity (using API profiles) .
• the Streptococcus 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 for use as a source of antigen.
• the organisms were boiled and washed in formed saline by repeated centrifugation, and they were then resuspended in 1% formol saline.
• 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 two intravenous injections after intervals of 1 and 2 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 80% T vaccine) intraperitoneally, 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.
• 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 FCS.
• PEG polyethylene glycol 1500
• 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.
• screening tests for the specific monoclonal antibody were made utilising the conventional enzyme immunoassay screening method described below.
• the clones were assayed by the enzyme immunoassay method to determine antibody production.
• 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 Streptococcus species and related genera bearing different antigens. Specifically, a grid of microtitre plates containing a representative selective of Streptococcus 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.
• mice were primed with pristane, for at least
• Purification was accomplished using the ammonium sulphate/DEAE method. Specifically, 10 ml of the ascites fluid were 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 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 M sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) . The solution was dialysed versus 2x1000 ml of the same buffer, at +4°C.
• 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.
• G. Enzyme-Monoclonal Linkage The monoclonal antibody specific against the antigen, prepared as above, 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 PBS pH 7.4, at +4°C.
• 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.
• glutaraldehyde was removed by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column, previously equilibrated in PBS.
• the conjugate was eluted with 3.5 ml PBS and then dialysed against 2 x 2000 ml of Tris buffer (50 mM Tris, 1 mM magnesium chloride, pH 8.0 plus 0.02% sodium azide) at +4°C.
• 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. If deemed necessary, 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.
• PVC polyvinyl chloride
• Example 1 The procedure of Example 1 was repeated, to give a monoclonal antibody broadly cross-reactive with each species of the genus Streptococcus.
• 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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