MONOCLONAL ANTIBODIES AND THEIR USE

Abstract: Monoclonal antibodies to the genus Escherichia, the labelled antibodies, compositions and kits containing them, and their use in diagnosis of antigen and treatment.

Description: MONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use. BACKGROUND OF THE INVENTION Among the species Escherichia, divisions have been made. The best known species is E . coli which will be used herein for the purposes of illustration. IS coli is described in Zinsser Microbiology (17th ed.) 734-5. There are somatic antigens of IS. coli which are defined herein by the labels 01 to 0159; capsular antigens designated Kl to K99; flagellar antigens designated Hi to H42; enterotoxins, i.e. heat-labile (LT) , heat-stable (ST) and vero toxin (VT) ; binding antigens, divided into two major factions, 1 and 2; endotoxins produced by 33. coli; and vero toxins which attack vero cells. IS. coli occupies a unigue position among opportunistic enteric bacilli in that certain strains are capable of causing primary intestinal disease as well as extra-intestinal infection. In addition, IS. coli has been the subject of more experimental research than any other micro-organism, especially in the field of molecular biology. IS. coli is the most common cause of urinary tract infection in man. It is also the most frequent cause of gram-negative sepsis and has been isolated from pneumonia, wounds and cerebrospinal fluid. 13. coli is a major cause of neonatal meningitis but it is rarely seen in older populations. The mortality in IS. coli meningitis is between 40 and 80% in new-borns, , and in survivors the majority have subsequent neurologic or developmental abnormalities. The invasiveness of E_. coli in this disease is apparently due to the capsular antigen K-l. —• col has also been associated with gastrointestinal disease in both man and animals. The enterpathogenicity of 33. coli appears to be mediated, either by production of an enterotoxin or by a shigella-like penetration of the intestinal mucosa. The toxin causes fluid accumulation in the jejunal and ileal portion of the intestine, while the E. coli that causes intestinal mucosal penetration resides primarily in the colon. Present treatment and diagnosis of E . coli infections vary depending on the locus of the infection. It is estimated that in the United States and Europe many millions of cases of bacterial diarrhea occur annually, of which several million are seen by a physician or admitted to a hospital. Because of the self-limiting nature of the adult disease, most people do not seek treatment. Of the people seeking treatment, bacterial diagnosis of diarrhea is presently made by stool culture techniques. These techniques are generally performed only in hospitals and are slow, requiring one to three days. During this time, the patient is exposed, if treated, to the expense and potential hazard of inappropriate therapy. However, if not treated, the patient is exposed to the hazard of a deteriorating condition pending the test result and initiation of therapy. 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. Presently, in urinary tract infections, 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. Accordingly, 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. Thus, existing methods of detection of 33. coli with high accuracy in urinary tract infections or gram-negative sepsis are less than satisfactory in that they consume large amounts of expensive skilled labour and laboratory time, generally taking one and often several days before returning results. The production of monoclonal antibodies is now a well-known procedure first described by Kohler and Milstein, Eur. J. Immunol. , 6_ (1975) 292. While the general technique of preparing hybridomas and the resultant monoclonal antibodies is understood, it has been found that preparing a specific monoclonal antibody to a specific antigen is difficult, mainly due to the degree of specificity and variations required in producing a particular hybridoma. SUMMARY OF THE INVENTION The present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of E. coli antigens and/or organisms. Briefly stated, the present invention comprises monoclonal antibodies specific for an antigen of IS. coli; in particular, the antigen or species of E. coli; the 01 to 0159 (inclusive) antigen or antigens of E_. coli; the Kl to K99 (inclusive) antigen or antigens of IS. coli; the Hi to H42 (inclusive) antigens of IS. coli; the binding antigens 1 and 2 of E. coli; the enterotoxins LTl of E. coli; the enterotoxins ST1 and ST2 of E_. coli; the endotoxins of E. coli; as well as a monoclonal antibody broadly cross-reactive with an antigen for each species (or substantially all species) of the genus E. coli. The invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Escherichia antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Escherichia 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 Escherichia antigens or organisms in a specimen, comprising contacting said specimen with the labelled monoclonal antibody in an appropriate immunoassay procedure. Additionally, the invention is also directed to a therapeutic composition comprising a monoclonal antibody for an antigen of Escherichia and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Escherichia. DETAILED DESCRIPTION The monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Escherichia 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 Escherichia 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 Escherichia 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 Escherichia 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 Escherichia antigen. The monoclonal antibody selected, which is specific for the particular Escherichia 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. Once the labelled monoclonal antibody is formed, 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. At the present time, enzyme immunoassays are preferred owing to their low cost, reagent stability, safety, sensitivity and ease of procedure. One example is the enzyme-linked immunosorbent assay (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 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. 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 comprising one or more of the monoclonal antibodies to the particular Escherichia 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 Escherichia 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 Escherichia in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Escherichia alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Escherichia 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, the incidence of significant diarrhea and diarrheal illness is so high that estimates of market size for such a kit are difficult to make, but a "same day" test could be expected to be used at least as often as stool cultures. Large use of such tests in developing countries might be anticipated because of more frequent and severe diarrhea, and other related illnesses. Additionally, a kit could be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis. Further, conjugated or labelled monoclonal antibodies for antigens, and/or species of Escherichia 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 Escherichia 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 Escherichia. One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Escherichia antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Escherichia antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Escherichia organisms in a particular sample. Monoclonal diagnostics which detect the presence of Escherichia antigens can also be used in periodic testing of water sources, food supplies and food processing operations. Thus, while 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 Escherichia 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 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. 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 SSI = Statenserum Institut, Copenhagen PHLS - Public Health Laboratory Service CVL = Central Veterinary Laboratory im = intramuscular ip = intraperitoneal iv = intravenous TSB = Tryptone Soya Broth CFA = Complete Freunds Adjuvant Example 1 A. Antigen Preparation Escherichia coli bearing an 08 antigen was obtained from the National Collection of Type Cultures (NCTC accession No. 9008) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . The Escherichia coli 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 saline by repeated centrifugation, and then resuspended in formol saline. B. Animal Immunisation Six Balb/c mice were injected with the prepared antigen, over five weeks. They were given one intraperitoneal injection per week for three weeks (0.1 ml 80% T vaccine) , followed by three intravenous injections, each after one week intervals, of LD- n of boiled killed Escherichia coli 08 antigen prepared as above. 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. C. Cell Fusion 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. In order to count the myeloma cells, 0.1 ml of the suspension was diluted to 1 ml and a haemacytometer with phase microscope was used. In order to count the spleen cells, 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 8 spleen cells were then mixed with 5 x 107 myeloma cells, the mixture washed in serum-free DMEM high in glucose, and centrifuged, and all the liquid removed.- 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. 10 μl of the mixture were placed in each of 480 wells of standard multiwe.ll tissue culture plates. Each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin and thymidine) and a feeder 4 layer of Balb/c macrophages at a concentration of 5 x 10 macrophages/well. 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. 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 Escherichia coli 08 antigen was developed in at least one well. D. Cloning From those wells which yielded antibody aginst the Escherichia coli 08 antigen, cells were removed and cloned by dilution culture. In limiting dilution, dilutions of cells suspensions in 18% FCS-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 clones were assayed by the enzyme immunoassay method to determine antibody production, and a positive clone recloned using the standard agar method. In the agar method, 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. An overlay of equal volumes of agar and cells in 18% FCS-DMEM was spread evenly over the base. The cells were allowed to multiply for approximately 10 days at 37 C, 7-9% CO., 95% 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. E. Monoclonal Selection The monoclonal antibodies from the clones were screened by the standard techniques for binding to Escherichia coli NCTC 9008, prepared as in the immunisation, and for specificity in a test battery of Escherichia coli and related species bearing different antigens. Specifically, a grid of microtitre plates containing a representative selective of 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. The monoclonals had the appropriate specificity (to the 08 antigen) , and were negative with respect to other E_. coli, Shigella, Salmonella and Serratia. • F. Antibody Production and Purification Balb/c mice were primed with pristane for at least 7 7 days, and were then 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 minutes, the cells discarded and the antibody-rich ascites collected and stored at -20 C. For purification, 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 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 Escherichia coli 08 antigen, prepared as above, was linked to an enzyme, viz. highly-purified alkaline phosphatase. 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 μl of a 20% glutaraldehyde in PBS solution added. The conjugation mixture was left at room temperature for 1.5 hours. After this time 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. The conjugate was then dialysed vs 2 x 2000 ml of TRIS buffer (50 mM TRIS, 1 mM magnesium chloride, pH 8.0 + 0.02% sodium azide) at +4 C. To the dialysed conjugate was added 1/lOth its own volume of 10% BSA in TRIS buffer. The conjugate was then sterile filtered through a 0.22 μm membrane filter into a sterile amber vial and stored at +4 C. H. Monoclonal Antibody Conjugate Testing 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. In this case, the monoclonal antibodies were found to be specific for the 08 antigen of Escherichia coli. The monoclonal antibodies were also tested and shown to be of the Class IgG2b. 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. Examples 2 to 42 The procedure as in Example 1 was followed in each of 4i cases, with differences outlined below, to prepare monoclonal antibodies and conjugates for various antigens of the genus Escherichia coli. The respective sources and strains of the antigens (with the number of the antigen indicated in brackets) were NCTC 9001 (01) , NCTC 11100 (02) , NCTC 9004 (04) , NCTC 9005 (05) , NCTC 11105 (06) , NCTC 9007 (07) , NCTC 9003 (03) , SSI A84a (09) , NCTC 9010 (010) , NCTC 9011 (011) , NCTC 9013 (013) , NCTC 9014 (014) , NCTC 9015 (015) , NCTC 9017 (017) , PHLS E20697/o (018) , NCTC 9020 (020) , NCTC 9021 (021) , NCTC 10249 (025) , NCTC 9027 (027) , NCTC 9028 (028), SSI 6181-66 (073) , NCTC 9078 (078) , CVL B41 (0101) , PHLS E482/o (0136) , PHLS E27048/O (0149) , PHLS E493/o/a (0152) , PHLS E14920/o/a (0153) , NCTC 10964 (0157) , LPS 017 (an endotoxin) , CS1 from Dublin (colonisation factor 1) , CS2 from Dublin (colonisation factor 2) , CS3 from Dublin (colonisation factor 3) , NCTC 11100. (HI) , NCTC 11100 (K2) , SSI Su 65/42 (K12) , SSI Su 4344-41 (K13) , Nobivac -(K88)*, CVL K99-B41 (K99) , LT from Dr. Richard A. Finkelstein (an exotoxin of human origin) , LT from Dr. Finkelstein (a labile exotoxin of human origin) , and P-LT from Dr. Finkelstein (a labile exotoxin of porcine origin) . In the antigen preparation step, the growth medium was DMEM in Examples 2, 7, 8, 9, 11, 15,, 16, 19, 20, 22, 24, 25, 26, 27, 28, 34, 35, 36, 37 and 39. The organisms were washed in saline in Examples 4, 7, 9, 10,22, 24, 36 and 39. Washing with saline was used in Examples 14, 18 and 21, boiling and washing with phenol saline in Examples 16, 19 and 25, and boiling and washing with formol saline in Example 17. In Examples 2, ' 8, 14, 15, 16, 19, 20, 25, 26, 27, 28, 34 and 35, resuspension was in phenol saline. The antigens used in Examples 30, 31, 32, 33, 37, 40, 41 and 42 were supplied in pure form. The animal immunisation procedure of Example 1 was modified by the addition of a further iv injection, after a further 25 weeks (Examples 2 and 39) , 16 weeks (Example 4) , 20 weeks (Example 8) , 3 weeks (Examples 32 and 34) or 8 weeks (Example 35) . Intrasplenic preparation was used in Examples 16, 25, 26, 27 and 28. Otherwise, injections and intervals (in weeks) were as follows: the sequence in Example 3 was ip-l-ip-l-ip-2-iv-2-iv-5-iv-4-iv-2-iv-2-iv- 2-iv-2-iv-2-iv; the sequence in Example 6 was ip-l-ip-l-ip-l-iv-l-iv-8-iv-2-iv-l-iv; the sequence in Example 7 was im (in CFA)-6-iv(in PBS) ; the sequence in Example 9 was ip-l-ip-l-ip-l-iv-2-iv-l-iv-3 days-iv; the sequence in Example 10 was ip-l-ip-2-ip-l-iv-l-iv-l-iv- 12-iv-2-iv; the sequence in Example 11 was im (in CFA)-9-iv(in PBS) ; the sequence in Example 14 was ip-l-ip-l-ip-l-iv-l-iv-4-iv; the sequence in Examples 15 and 19 was im (in CFA)-5-iv(in PBS) ; the sequence in Example 17 was ip-l-ip-l-ip-l-iv-l-iv-2-iv; the sequence in Example 21 was ip-l-ip-l-ip-l-iv-l-iv-2-iv-2-iv-l-iv- l-iv-2-iv; the ' sequence in Example 23 was ip-l-ip-l-ip- l-iv-4-iv-l-iv-3-iv-2-iv-2-iv-2-iv-8-iv; the sequence in Example 24 was ip-2-ip-2-ip-l-iv-l-iv-l-iv-7-iv-l-iv; the sequence in Example 30 was ip-1-ip-l-ip-l-iv-l-iv; the sequence in Example 31 was im (CFA) -3-ip(in PBS) -3- ip(in PBS)-4-iv; the sequence in Examples 32 and 33 was im (in CFA) -4-iv; the sequence in Examples 36 and 37 was ip-2-ip-l-ip-l-iv-l-iv-l-iv-l-iv; the sequence in Example 38 was ip-l-ip-l-ip-2-iv-l-ip; the sequence in Examples 40 and 41 was ip-l-ip-l-ip-2-iv-4-iv; and the sequence in Example 42 was ip-l-ip-l-ip-l-iv-2-iv-l-iv-3-iv. 7 In the cell fusion step, 6 x 10 myeloma cells were used in Examples 3, 8, 9, 20, 22, 23, 24, 33, 34, 35, 40, 7 7 41 and 42, 10 in Example 6, 3 x 10 in Example 21, and 7 7 8 x 10 in Example 30. 2 x 10 spleen cells were used in Example 3, 1.4 x 10 in Examples, 17, 22, 34 and 35, 1.5 8 7 x 10 in Examples 24, 40 and 41, 5.4 x 10 in Example 30, and 1.44 x 10 in Example 42. In the cloning step, the standard agar method was used alone in Examples 3, 5, 21, 23 and 30. Both limiting dilution and agar methods were used in Examples 12, 14, 17, 18, 29, 40 and 41. In the antibody production step for Examples 7, 8, 9, 10, 11, 13, 16, 18, 19, 25, 26, 27 (in addition to the technique given for Example 1), 28, 34, 35, 36 and 38, the following procedure was used: Cells of the monoclonal antibody-producing cell line were grown in batch tissue culture. DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume. The culture was then allowed to overgrow, to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected. In the antibody purification step for Examples 3, 4, 5, 12/ 14, 17, 20, 23, 25, 40 and 41, 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.lM 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 of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (O.lM sodium citrate, pH 3.5) into sufficient IM 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. In addition to the technique of Example 1, antibody purification for Example 8 was conducted as follows: To one litre of culture supernatant was added one litre of 0.05M sodium acetate buffer, pH 4.5, and 40 ml of SP-Sephadex, previously equilibrated in O.lM sodium acetate buffer, pH 5.0. The suspension was stirred at +4 C for one hour. The SP-Sephadex was allowed to settle and the supernatant decanted. The SP-Sephadex was packed in a column, washed with 60 ml of O.lM acetate buffer, pH 5.0, and eluted with 60 ml of the same buffer plus IM sodium chloride. The eluate was stirred at +4 C, and an equal volume of saturated ammonium sulphate added slowly. 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. The antibody purification step for Examples 9, 13, 25, 34, 35, 36 and 38 was conducted as follows: To one 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/min to a 1 ml column of Protein A-Sepharose, previously equilibrated with O.lM TRIS buffer, pH 8.2. The column was then washed with 40 ml of O.lM TRIS buffer. The monoclonal antibody was eluted with citrate buffer (O.lM sodium citrate, pH 3.5) into sufficient IM 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. The antibody purification step for Examples 10, 16, 26 and 28 was conducted as follows: 500 ml tissue culture supernatant were concentrated ' to about 50 ml on an Amicon XM300 ultrafiltration membrane. The concentrate was diluted to 500 ml with 0.9% sodium chloride and reconcentrated to about 50 ml. The concentrate was stirred at +4 C, and an equal volume of saturated ammonium sulphate added slowly. The suspension was stirred for a further 30 minutes. The precipitate was then harvested by centrifugation at 10,000 g for 10 minutes. The precipitate was dissolved in a minimum volume of cold TRIS/acetic acid buffer (O.lM TRIS/acetic acid + 0.02% sodium azide, pH 7.5) and dialysed versus 2 x 1000 ml of the same buffer at +4 C. The redissolved, dialysed precipitate was centrifuged at 30,000 g for 30 minutes, filtered through a 0.45 μm membrane filter and applied to a 215 mm x 300 mm TSK G 3000SW gel filtration column. The monoclonal antibody was eluted with TRIS/acetic acid buffer and concentrated by vacuum dialysis. The antibody purification step for Examples 21, 27 and 29 was conducted as follows: Ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was diluted with 9 times its own volume of cold PBS and stirred at -4 C. An equal volume of cold, saturated ammonium sulphate was added slowly. The mixture was stirred for a further 30 minutes after 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 TRIS-acetate buffer (O.lM TRIS pH 7.5 with glacial acetic acid + 0.02% sodium azide). The solution was dialysed versus 2 x 1000 ml of the same buffer at +4 C. The dialysed, redissolved precipitate (5.4 ml) was centrifuged at 30,000 g for 20 minutes ' then filtered through a 0.45 μm membrane filter. A portion of the filtrate (1.0 ml) was applied to a 21.5 mm x 300 mm TSK G-3000SW gel filtration column previously equilibrated in TRIS-acetate buffer. The monoclonal antibody was eluted in TRIS-acetate buffer. Antibody conjugation in Examples 10, 17, 21, 25, 26, 28, 34 and 42 was by the benzoquinone method. 24 mg 5 alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 300 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 1C 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 J5 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 ?c phosphatase and immediately followed by sufficient IM sodium bicarbonate to give a final concentration of O.lM. 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 O.lM. 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 μm membrane filter into a sterile amber vial, and stored at +4 C. 30 On selection at least, the monoclonals exhibited the appropriate specificity and, where it could be established, were negative with respect to other E_. coli. It may be noted that the monoclonal of Example 3 was specific to NCTC 9002 and 11100 both bearing the 02 35 antigen; the monoclonal of Example 6 was specific to IS. coli 06 (NCTC 11105, 9006); the monoclonal of Example 20 was specific to E. coli strains 9027 and E18535 both bearing the 027 antigen; the monoclonal of Example 29 was specific to E. coli strains 10964 and E32157/o both bearing the 0157 antigen; the monoclonal of Example 32 was specific to those strains of 33. coli bearing the CS2 antigen, C19f, 201 74 B34334f; the monoclonal of Example 33 was specific to 13. coli strains C9216-1, 201 74, B34212b, 34334f which ali bear the CS3 antigen; the monoclonal of Example 34 was specific to IS. coli strains NCTC 11100, 9006, 9022 and 10430 which all bear the HI antigen; the monoclonal of Example 38 showed a strong reaction against all sub-units of IS. coli K88; the monoclonal of Example 40 is specific to E_. coli labile endotoxin of human and porcine origin, and probably recognises the alpha-subunit of the protein; and the monoclonal of Example 41 is possibly specific to the beta-subunit of the E. coli LT of human origin. Negative reactions were obtained with respect to Klebsiella, Pseudomonas, Enterobacter, Shigella and Salmonella for the monoclonals of Examples 3, 4, 5, 6, 8, 9, 10, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, 25, 26, 29, 30, 32, 34, 38, 39, 40, 41 and 42, with the proviso that there was no test for Klebsiella in Examples 10, 16, 34 and 41, Pseudomonas in Examples 10, 16, 23 and 39, Enterobacter in Examples 4, 13, 20, 25, 30, 34, 39 and 41, Shigella in Examples 3 and 20, and Salmonella in Example 3. Further, the monoclonals of Examples 8, 14, 18, 23, 29 and 38 were negative with respect to Serratia, the monoclonal of Example 8 was negative with respect to Campylobacter, the monoclonals of Examples 20 and 21 were negative with respect to Streptococcus, the monoclonals of Examples 30 and 41 were negative with respect to Citrobacter, and the monoclonals of Examples 40, 41 and 42 were negative with respect to cholera toxin. The Sub-class was IgM for Examples 2, 10, 16, 18, 21, 26, 27, 28 and 29, IgG3 for Examples 3, 4, 5, 9, 11, 12, 13, 14, 19, 20, 23, 30 and 36, IgG2a for Examples 6, 19, 24, 35 and 38, and IgGl for Examples 8, 17, 22, 32, 39, 40, 41 and 42. The monoclonals of Examples 7 and 37 were positive with Protein A, indicating the Sαb-class IgG2a, IgG2b or IgG3. Example 43 The general procedure of Example 1 is used in preparing a monoclonal antibody broadly cross-reactive with antigens of the Escherichia genus. 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.

Claims: WHAT IS CLAIMED IS: 1. A monoclonal antibody specific for an antigen or species of Escherichia. 2. The antibody of Claim 1 specific to the antigen or species of Escherichia coli. 3, The antibody of Claim 1 specific to the 01 to 0159 (inclusive) antigen or antigens of Ea herichia coli . 4. The antibody of Claim 1 specific to the 08 antigen of Escherichia coli. 5. The antibody of Claim 1 specific to the Kl to K99 (inclusive) antigen or antigens of Escherichia coli. 6. The antibody of Claim 1 specific to the Hi to H42 (inclusive) antigen or antigens of Escherichia coli. 7. The antibody of Claim 1 specific to the enterotoxins of Escherichia coli . S. The antibody of Claim 1 specific to the heat labile (LT) enterotoxins of Escherichia coli. 9. The antibody of Claim 1 specific to the heat labile LTl enterotoxins of Escherichia coli. 10. The antibody of Claim 1 specific to the heat labile LT2 enterotoxins of Escherichia coli. 11. The antibody of Claim 1 specific to the heat stable (ST) enterotoxins of Escherichia coli. 12. The antibody of Claim 1 specific to the heat stable ST1 enterotoxins of Escherichia coli. 13. The antibody of Claim 1 specific to the heat stable ST2 enterotoxins of Escherichia coli. 14. The antibody of Claim 1 specific to endotoxins of Escherichia coli. _ _ 15. The antibody of Claim 1 specific to one or more epitopic sites on endotoxins of Escherichia coli. 16. The antibody of Claim 1 specific to -* binding antigen of Escherichia coli. 17. The antibody of Claim 1 specific to binding antigen 1 of Escherichia coli. 18. The antibody of Claim 1 specific to binding antigen 2 of Escherichia coli. 10 19. A monoclonal antibody broadly cross- reactive with an antigen of all species of the genus Escherichia. 20. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 151-19 and an appropriate label. 21. The labeled monoclonal antibody of Claim 20, wherein said label is a member of the group selected from a radioactive isotope, enzyme,. fluorescent compound, bioluminescent 20 compound, chemiluminescent compound, or ferro- magnetic atom, or particle. 22. The labeled monoclonal antibody of Claim 21, wherein said label is an enzyme capable of conjugating with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure. 23. The labeled monoclonal antibody of Claim 22, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase. » 24. The labeled monoclonal antibody of Claim 21, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluorescent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immunoassay, photon counting immunoassay, or the like procedure. 25. The labeled monoclonal antibody of Claim 24, wherein said fluorescent compound or probe is fluorescein. 26. The labeled monoclonal antibody of Claim 21, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay. 27. The labeled monoclonal antibody of Claim 26, wherein such chemiluminescent compound is luminol or a luminol derivative. 28. The labeled monoclonal antibody of Claim 21, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay. 29. The labeled monoclonal antibody of Claim ' 28, wherein such bioluminescent compound is luciferase or a luciferase derivative. 30. A process for diagnosing for the pre- sence of an antigen of Escherichia in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 20 in an immunoassay procedure appropri¬ ate for said label. 31. The process of Claim 30, wherein the appropriately labeled immunoassay procedure is selected from immuno-fluorescent or fluorescent immunoassay, immuno-electron microscopy, radio- metric assay systems, enzyme-linked immunoassays, fluorescence polarization, photon-counting bio- luminescent, or chemiluminescent immunoassay. 32. The process of Claim 31, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure. 33. The process of Claim 32, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase. 34. The process of Claim 31, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluores- cent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immuno¬ assay, or photon-counting immunoassay, or the like procedure. 35. The process of Claim 34, wherein said fluorescent compound or probe is fluorescein. 36. The process of Claim 31, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay. 37. The process of Claim 36, wherein said chemiluminescent compound is luminol or a luminol derivative. 38. The process of Claim 31, wherein said label is a bioluminescent compound capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay. 39. The process of Claim 38, wherein said bioluminescent compound is luciferase or a lucif- erase derivative. 40. A therapeutic composition comprising one or more of the monoclonal antibodies of Claims 1-19 and a pharmaceutically acceptable carrier or diluent. 41. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 20 and a pharmaceutically acceptable carrier or diluent. 42. A method of treating Escherichia infec¬ tions comprising administering an effective amount of a monoclonal antibody of Claims 1-19. 43. A kit for diagnosing for the presence of an antigen or species of Escherichia in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-19. 44. The kit of Claim 43, wherein said at least one antibody is labeled. 45. The kit of Claim 44, wherein said at least one monoclonal antibody is labeled with a fluorescent compound. 46. The kit as in Claim 44, wherein said at least one monoclonal antibody is labeled with an enzyme. 47. The kit as in Claim 44, wherein said at least one monoclonal antibody is labeled with a member of the group consisting of a radio- active isotope, chemiluminescent compound, bio¬ luminescent compound, ferromagnetic atom, or particle . 48. The kit of Claims 44, 45, 46, and 47 additionally containing at least one known Escherichia antigen as a control. 49. The kit of Claims 44, 45, 46, 47, and 48 containing each known antigen of Escheri¬ chia. 50. The kit of Claims 44, 45-, 46, 47, and 48 containing the antigen or antigens of 0 Escherichia coli. 51. A kit for diagnosing for the presence of an antigen or species of Escherichia in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-19 and a control. *L5 52. The kit of Claim 51, wherein said at least one antigen is labeled and said control is at least one known antigen of Escherichia. 53. A kit for diagnosing for the presence of a gram-negative bacterial infection comprising 20 at least one monoclonal antibody of Claims 1-19. 54. The kit of Claim 53, wherein said at least one monoclonal antibody is labeled.

Inventors: COX PETER JOHN, NOYES ALICE MARGARET, WIDDOWS DANNY, WRIGHT BRUCE WILLIAM, COX, PETER, JOHN, NOYES, ALICE, MARGARET, WIDDOWS, DANNY, WRIGHT, BRUCE, WILLIAM
Applicants: TECHNOLOGY LICENCE CO LTD
Application year: 1985
Main classification: C07K15
IPC codes: A61K38, A61K39, C07K1, C07K14, C07K16, C07K19, C12N15, C12P21, C12R1, G01N33