EP0192725A1 - Monoclonal antibodies and their use - Google Patents

Abstract

Monoclonal antibodies for the genus Enterobacter, labeled antibodies, compositions and kits containing them, and their use for diagnosis of antigens and treatment.

Description

MONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use.

BACKGROUND OF THE INVENTION Enterobacter is described in Zinsser Microbiology (17th ed.) 729-30. The clinical importance of the genus Enterobacter as a separate entity was not greatly appreciated until the 1960's. Prior to this time separation of the Enterobacter from the Klebsiella was not routinely attempted, and many infections were reported as being caused by the Klebsiella-Aerobacter group. How many of these diseases were in reality caused by Enterobacter is not known. Recent studies indicate that Enterobacter infections occur less frequently than those caused by Klebsiella.

Divisions have been made among the Enterobacter species. The more commonly known Enterobacter members include Enterobacter aerogenes, Enterobacter cloacae and Enterobacter agglomerans. The somatic _erotypes of Enterobacter are labelled 01 to 028, while the capsular serotypes have not been classified.

The serotyping of Enterobacter is not uniform among medical scientists. The system adopted herein is the Pitt system, as defined by T. L. Pitt, Division of Hospital Infections, Central Public Health Laboratories, Colindale Avenue, London N 9 5HT, England.

Enterobacter, like most Enterobacteriaceae, are capable of producing disease in any body tissue, but have been most frequently isolated from urinary tract infections. Enterobacter cloacae accounts for the majority of clinical isolates of this genus, but all species have been isolated from clinical specimens. Two species, E_. agglomerans (formerly classified as Erwinia) and E. cloacae, were associated with an epidemic throughout the USA, involving contaminated intravenous fluids. These species were isolated from eight hospitals in seven of the United States, and were responsible for

150 bacteremias and nine deaths. An enterotoxin-producing E. cloacae has been isolated from the jejunal aspirate of a patient with tropical sprue.

This strain produced a toxin similar to the ST toxin of

E. coli. As with the enterotoxigenic Klebsiella isolates, the exact role of Enterobacter in intestinal disease has not been fully explored. Other Enterobacter species can be isolated from clincal material, but with less frequency than E. cloacae, Enterobacter sakazakii was first isolated from an infant with neonatal meningitis. Enterobacter gergoviae has been implicated in nosocomial urinary tract infections and has been isolated from wounds, sputum and blood. Enterobacter aerogenes and E_. hafniae cause similar infections.

With the exceptions of ampicillin and cephalosporins, most of the anti-microbials are useful in the treatment of Enterobacter infections. As with all enterics, resistance patterns may vary with individual isolates.

Enterobacter 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.

Enterobacter is also known to cause urinary tract infection. Urinary tract infection symptoms include fever, pain in the back or abdomen, and frequency of urination. Gram-negative sepsis can occur during the course of a urinary tract infection.

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 Enterobacter 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. (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 Enterobacter antigens and/or organisms. Briefly stated, the present invention comprises monoclonal antibodies specific for an antigen of Enterobacter; in particular, the antigen or antigens of Enterobacter aerogenes, the antigen or antigens of Enterobacter cloacae, the antigen or antigens of

Enterobacter agglomerans, the 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, 013, 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027 and 028 antigens, and also the capsular antigens,of Enterobacter, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species (or substantially all species) of the genus Enterobacter.

The invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Enterobacter antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Enterobacter 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 Enterobacter 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 Enterobacter and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Enterobacter.

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 Enterobacter 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 Enterobacter 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 Enterobacter 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 Enterobacter 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 Enterobacter antigen. The monoclonal antibody selected, which is specific for the particular Enterobacter 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 jLn 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 Enterobacter 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 Enterobacter 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

Enterobacter in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Enterobacter^'alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Enterobacter 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 Enterobacter 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 Enterobacter 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 Enterobacter.

One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Enterobacter antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Enterobacter antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Enterobacter organisms in a particular sample.

Monoclonal diagnostics which detect the presence of Enterobacter 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 Enterobacter 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 ip = intraperitoneal iv = intravenous Example 1 A. Antigen Preparation

Enterobacter aerogenes antigen (epitope 2) was obtained from the National Collection of Type Cultures (NCTC accession No. 10006) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . The

Enterobacter aerogenes 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 DMEM and harvested. The organisms were boiled and washed in formol saline by repeated centrifugation, and then resuspended in formol saline. B. Animal Immunisation

Balb/c mice were injected with the prepared antigen. They were given one ip injection per week for three weeks (0.05 ml 80% T vaccine), followed by an iv injection each week for three weeks of Enterobacter aerogenes vaccine prepared as above, followed by a further iv injection after a 4 week interval. 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 NSO (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 6 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 672 wells of standard multiwell 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% 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 Enterobacter aerogenes antigen was developed in at least one well.

D. Cloning From those wells which yielded antibody aginst the Enterobacter aerogenes antigen, cells were removed and cloned using the standard agar method. In the agar method, a freshly-prepared stock solution of sterile 1.2% 5 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

10 evenly over the base. The cells were allowed to multiply for approximately 10 days at 37 C, 7-9% C0₂, 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

15 supernatants were assayed for specific antibody by the standard enzyme immunosorbent assay.

The clones were assayed by the enzyme immunoassay method to determine antibody production.

E. Monoclonal Selection

20 The monoclonal antibodies from the clones were screened by the standard techniques for binding to Enterobacter aerogenes NCTC 10006, prepared as in the immunisation, and for specificity in a test battery of Enterobacter aerogenes species and related genera bearing

25 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.

30 The appropriate specificity (to NCTC 10006) was observed. The monoclonal was negative to other Enterobacter, Salmonella, Shigella, E. coli and Serratia.

F. Antibody Production and Purification

Balb/c mice were primed with pristane for at least 7 ,, 7

-i-¹ 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 Enterobacter aerogenes antigen, prepared as above, was linked to an enzyme, viz. highly-purified alkaline phosphatase. The one-step glutaraldehyde method and benzoquinone conjugation were both used.

In 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 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 l/10th 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. In the benzoquinone method, 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25 M sodium phosphate buffer, pH 6.0, at +4 C. 18 mg p-benzoquinone were dissolved in 0.6 ml warm AR ethanol, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. Unreacted benzoquinone and reaction by-products were then .removed and the buffer exchanged by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column previously equilibrated in 0.15M sodium chloride. The benzoquinone-activated alkaline phosphatase thus produced was sufficient for six 1.5 mg antibody conjugations. Monoclonal antibody was dialysed against 2 x 500 ml of 0.15M sodium chloride at +4 C. Dialysed antibody was added to 4 mg of benzoquinone-activated alkaline phosphatase and immediately followed by sufficient IM sodium bicarbonate to give a final concentration of 0.1M. The conjugation mixture was left in the dark at +4 C for 48 hours. Sufficient IM lysine was then added to give a final concentration of 0.1M. After 2 hours in the dark at room temperature, the conjugate was dialysed against 2 x 1000 ml PBS + 0.02% sodium azide at +4 C. An equal volume of glycerol was added. The conjugate was sterile-filtered through a 0.22 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 antigen Enterobacter aerogenes. The monoclonal antibodies were tested and shown to be of the Subclass IgGl.

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 13 The procedure of Example 1 was followed in each of

12 cases, with differences outlined below, to prepare monoclonal antibodies and conjugates for various antigens of the genus Enterobacter.

In Example 2, the antigen was Enterobacter aerogenes (epitope 1) , NCTC 10006; in Examples 3 and 4, respectively epitopes 1 and 2 of Enterobacter cloacae,

NCTC 10005; in Examples 5 to 12, Enterobacter cloacae respectively bearing the antigens 03 (NCTC 11572) , 07

(NCTC 11576) , 010 (NCTC 11579) , 011 (NCTC 11580) , 014 (NCTC 11583) , 015 (NCTC 11584) , 019 (NCTC 11588) and 025

(NCTC 11594) ; and, in Example 13, an alpha antigen of

Enterobacteriaceae, Klebsiella NCTC 9137.

In the antigen preparation step for Example 5, the organisms were boiled and washed once and resuspended in phenol saline. In Examples 6, 7 and 8, the organisms were boiled and washed in saline, and resuspended in phenol saline. In Examples 9, 10, 11 and 12, the organisms were boiled and washed in phenol saline, and resuspended in phenol saline. In Example 13, the organisms were washed in formol saline and resuspended in formol saline.

In the animal immunisation step for Examples 5, 8 and 13, the 4 week interval before the last iv injection was changed to 40, 1 and 6 weeks, respectively. In Examples 9, 10, 11 and 12, immunisation was intrasplenic.

7 In the cell fusion step, 5 x 10 myeloma cells were used in Examples 5, 6, 7, 8, 9, 10, 11 and 12.

Additionally for Example 3 , and as the cloning method in Examples 5, 6, 7, 8, 9, 10, 11 and 12, the dilution method was used. 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% C0₂ until semi-confluent. The supernatants were then assayed for specific antibody by the standard enzyme immunosorbent assay.

Additionally in Example 2, antigen purification 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 0.IM 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 0.IM 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. in Examples 3, 4, 6, 7 and 8, antibody purification was conducted as follows:

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 (0.1M 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 (0.1M 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 Example 9, antibody purification 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 0.1M TRIS buffer, pH 8.2. The column was then washed with 40 ml of 0.1M TRIS buffer. The monoclonal antibody was eluted with citrate buffer (0.1M 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 Example 12, antibody purification 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 (0.1M 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.

The glutaraldehyde method was used for conjugation in Examples 2, 7, 8, 9 and 13. The benzoquinone method was used in Examples 3 and 4. on selection at least, the appropriate specificity was shown in Examples 5, 6, 7, 8, 9, 10, 11 and 12, and the monoclonals of these Examples and of Examples 2 and 9 were negative with respect to other Enterobacter. Cross-reactivity was observed in Example 2 (with NCTC 10006, 10336 and 9735) , Example 3 (with various

Enterobacter cloacae NCTC 8168, 9394, 9395, 9396, 9736, 9785, 10005, 11570, 11571, 11572, 11573, 11575, 11576, 11577, 11584, 11585, 11586, 11587, 11588, 11590, 11591, 11593, 11594, 11595 and 11596) , Example 4 (with Enterobacter cloacae NCTC 9394, 9396, 10005, 11570, 11571, 11572, 11573, 11574, 11575, 11576, 11577, 11585, 11586, 11587, 11588, 11590, 11591, 11593, 11594, 11595 and 11596) and Example 13 (with Klebsiella NCTC 9660, 9128, 9129, 9137, Providencia NCTC 2481, 6932, 6933, 6934, 8113 and Hafnia alvei NCTC 8535) .

The monoclonals of Examples 1, 2 , 4 , 6 , 8, 11 and 13 were negative to Salmonella, of Examples 1, 2, 4, 8, 11 and 13 to Shigella, of Examples 1, 2, 4, 6, 7, 8, 9, 10, 11 and 13 to E_. coli, of Examples 1, 2, 6, 7, 8, 10, 11 and 12 to Serratia, of Examples 4, 6, 7, 8, 10, 12 and 13 to Pseudomonas, of Examples 4, 6, 8, 12 and 13 to Klebsiella, of Example 3 to many antigens other than Enterobacter, and of Example 13 to Citrobacter, Campylobacter, Proteus and Providencia. The Subclass IgG2a was found for Examples 3, 5, 7 and 13, IgG3 for Examples 4, 6 and 9, IgG2b for Example 8, and IgM for* Examples 10, 11 and 12.

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 Enterobacter.

2. The antibody of Claim 1 specific to the antigen of Enterobacter aerogenes.

3. The antibody of Claim 1 specific to the antigen of Enterobacter cloacae.

4. The antibody of Claim 1 specific to the antigen of Enterobacter agglomerans.

5. The antibody of ^• Claim 1 specific to the 01 antigen of Enterobacter.

6. The antibody of Claim 1 specific to the 02 antigen of Enterobacter.

7. The antibody of Claim 1 specific to the 03 antigen of Enterobacter.

8. The antibody of Claim 1 specific to the 04 antigen of Enterobacter.

9. The antibody of Claim 1 specific to the 05 antigen of Enterobacter.

10. The antibody of Claim 1 specific to the 06 antigen of Enterobacter.

11. The antibody of Claim 1 specific to the 07 antigen of Enterobacter.

12. The antibody of Claim 1 specific to the 08 antigen of Enterobacter.

13. The antibody of Claim 1 specific to the 09 antigen of Enterobacter.

14. The antibody of Claim 1 specific to the O10 antigen of Enterobacter.

15. The antibody of Claim 1 specific to the Oil antigen of Enterobacter.

16. The antibody of Claim 1 specific to the 012 antigen of Enterobacter.

17. The antibody of Claim^' 1 specific to the

013 antigen of Enterobacter.

18. The antibody of Claim 1 specific to the 014 antigen of Enterobacter.

19. The antibody of Claim 1 specific to the 015 antigen of Enterobacter.

20. The antibody of Claim 1 specific to the 016 antigen of Enterobacter.

21. The antibody of Claim 1 specific to the 017 antigen of Enterobacter.

22. The antibody of Claim 1 specific to the 018 antigen of Enterobacter.

23. The antibody of Claim 1 specific to the 019 antigen of Enterobacter.

24. The antibody of Claim 1 specific to the 020 antigen of Enterobacter.

25. The antibody of Claim 1 specific to the 021 antigen of Enterobacter.

26. The antibody of Claim 1 specific to the 022 antigen of Enterobacter.

27. The antibody of claim 1 specific to the 023 antigen of Enterobacter.

28. The antibody of claim 1 specific to the 024 antigen of Enterobacter.

5 29. The antibody of claim 1 specific to the 025 antigen of Enterobacter.

30. The antibody of claim 1 specific to the 026 antigen of Enterobacter.

31. The antibody of claim 1 specific to the 027 antigen lO of Enterobacter.

32. The antibody of claim 1 specific to the 028 antigen of Enterobacter.

33. The antibody of any of claims 5 to 32, specific to Enterobacter cloacae.

" 34. The antibody of claim 1 specific to a capsular antigen of Enterobacter.

35. A monoclonal antibody broadly cross-reactive with an antigen of all species of the genus Enterobacter.

36. A labelled monoclonal antibody consisting

20 essentially of a monoclonal antibody of any preceding claim and an appropriate label.

37. The labelled monoclonal antibody of claim 36, wherein said label is a member of the group selected from radioactive isotopes, enzymes, fluorescent compounds,

25 bioluminescent compounds, chemiluminescent compounds, ferromagnetic atoms and particles.

_ _.

38. The labelled monoclonal antibody of

Claim 37, wherein said label is an enzyme capable of conjugating with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure.

39. The labeled monoclonal antibody of Claim 38, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase.

4C_ The labeled monoclonal antibody of Claim 37, 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.

41. The labeled monoclonal antibody of Claim 40 , wherein said fluorescent compound or probe.is fluorescein.

42. The labeled monoclonal antibody of Claim 37, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.

43. The labeled monoclonal antibody of Claim 42, wherein such chemiluminescent compound is luminol or a luminol derivative.

44. The labeled monoclonal antibody of Claim 37 , wherein said label is a bioluminescent compound capable . of being used in an appropriate bioluminescent immunoassay.

45. The labeled monoclonal antibody of Claim 44 , wherein such bioluminescent compound is luciferase or a luciferase derivative.

46 . A process for diagnosing for the pre¬ sence of an antigen of Enterobacter in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 36. in an immunoassay procedure appropri¬ ate for said label.

47, The process of Claim 46, 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.

48. The process of Claim 47 , wherein said label is an enzyme capable of being used in an enzyme-linked immunoassa.y procedure.

49. The process of Claim 48 , wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase.

50. The process of Claim 47 , 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.

51. The process of Claim 50, wherein said fluorescent compound^"or probe is fluorescein.

52. The process of Claim 47, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.

53. The process of Claim 52 , wherein said chemiluminescent compound is luminol or a luminol derivative.

54^*. The process of Claim 47 , • wherein said label is a bioluminescent compound 'capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay.

55. The process of Claim 54 , wherein said bioluminescent compound is luciferase or a lucif- erase derivative..

56. A therapeutic composition comprising one or more of the monoclonal antibodies of Claims 1 to 35 and a pharmaceutically acceptable carrier or diluent.

57. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 36^' and a pharmaceutically acceptable carrier or diluent.

58. A method of treating Enterobacter infection comprising administering an effective amount of a monoclonal antibody of any of claims 1 to 35.

59. A kit for diagnosing for the presence of an antigen or species of Enterobacter in a diagnostic specimen comprising at least one monoclonal antibody of C_aims 1-35.

60.. The kit of Claim 59, wherein said at least one antibody is labeled.

61. The kit of Claim 60 , wherein said at least one monoclonal antibody is labeled with a fluorescent compound.

62. The kit as in Claim 60 , wherein said at least one monoclonal antibody is labeled with an enzyme.

63. The kit as in Claim 60 , 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.

64. The kit of any of claims 60 to 63 additionally containing at least one known Enterobacter antigen as a control.

65. The kit of any of claims 60 to 64 containing each known antigen of Enterobacter aerogenes, Enterobacter cloacae and Enterobacter agglomerans.

66. The kit of any of claims 60 to 64 containing the 01 to 028 antigens of Enterobacter.

67. The kit of claim 66, wherein the Enterobcater is

Ϊ0^" Enterobacter cloacae.

68. The kit of any of claims 60 to 64, containing a capsular antigen of Enterobacter.

69. A kit for diagnosing for the presence of an antigen or species of Enterobacter in a diagnostic specimen,

15 comprising at least one monoclonal antibody of claims 1 to 35 and a control.

70. The kit of claim 69, wherein said at least one antigen is labelled and said -control is at least one known antigen of Enterobacter.

20

71. A kit for diagnosing for the presence of a gram-negative bacterials infection comprising at least one monoclonal antibody of claims 1 to 35.

72. The kit of claim 71, wherein said at least one monoclonal antibody is labelled.

25

30

35