WO1986000646A1

WO1986000646A1 - Monoclonal antibodies and their use

Info

Publication number: WO1986000646A1
Application number: PCT/GB1985/000296
Authority: WO
Inventors: Bruce William Wright; Peter John Cox; Alice Margaret Noyes; Danny Widdows
Original assignee: Technology Licence Company Limited
Priority date: 1984-07-03
Filing date: 1985-07-02
Publication date: 1986-01-30
Also published as: EP0189451A1; JPS61502632A; GB8416845D0

Abstract

Monoclonal antibodies to the genus Shigella, 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 Shigella is described in Zinsser Microbiology (17th ed.) 739-741, as primarily human pathogens, but they are also isolated occasionally from other animals. The genus Shigella is classified in the tribe Escherichieae, and Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei constitute the four species of the genus. Speciation is based upon serologic and biochemical reactions.

Highly specific divisions have been among the Shigella species on the basis of antigenic structures. In general, all Shigella possess 0 antigens, while only some possess K antigens. The Shigellae may be divided into four major 0 antigenic groups designated A, B, C and D, which correspond to the species S_. dysenteriae, S_. flexneri, S_. boydii and S_. sonnei, respectively. Each major group or species is also sub-divided into types on the basis of the 0 antigen. These sub-groups are designated by arabic numbers. At the present time, there are 10 serologic types of S_. dysenteriae, 6 of S_. flexneri, 15 of S_. boydii and 1 of i3. sonnei described. The plethora of gram-negative bacilli and the wide variety of serogrouping or serotyping systems that are ^•available for organisms such as Shigella reflect the formidable task involved in defining the epidemiology of certain infections. However, the extreme specificity of antigen-antibody reactions has made it possible to recognise differences between strains of a bacterial species that are indistinguishable on the basis of other phenotypic criteria. All four species can cause bacillary dysentery, but the severity of disease, mortality and frequency of isolation differs for each species.

The members of the genus are the agents of a specifically human enteric disease, bacterial dysentery. Bacterial diarrhea is a common and often serious condition manifest as fluid loss from the bowel, leading in many cases to dehydration, and occasionally death.

In addition to diarrhea, Shigella 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. Present treatment and diagnosis of Shigella 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. Thus, existing methods of detection of Shigella with high accuracy in diarrheal 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 Shigella antigens and/or organisms. Briefly stated, the present invention comprises monoclonal antibodies specific for an antigen of Shigella; in particular, the 0A1, OA2, OA3, OA4, OA5, OA6, OA7, OA8, OA9 and OA10 antigens of Shigella dysenteriae, the OBl,.OB2, OB3, OB4, OB5 and OB6 antigens of Shigella flexneri, the OCl, OC2, OC3, OC4, OC5, OC6, OC7, OC8, OC9, OC10, OCll, OC12, OC13, OC14 and OC15 antigens of Shigella boydii, and the ODl antigen of Shigella sonnei, the invasiveness antigens 1 and 2, attachment antigens 1 and 2, enterotoxin 1 and 2, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Shigella.

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

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 Shigella 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 Shigella antigen chosen, generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the appropriate Shigella 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 Shigella 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 Shigella antigen. The monoclonal antibody selected, which is specific for the particular Shigella 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 Shigella 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 shigellosis and they are used in amounts effective to cure; the amount may vary widely, depending upon the individual being treated and the severity of the infection.

One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens or species of Shigella in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Shigella alone or as part of a kit i containing antibodies that can identify other bacterial genera or species of Shigella and/or other bacteria.

In the past, there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens; e.g. faeces 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, conjugated or labelled monoclonal antibodies for antigens and/or species of Shigella 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

Shigella 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 Shigella.

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

Monoclonal diagnostics which detect the presence of Shigella 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 Shigella 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 Example 1 A. Antigen Preparation

Shigella boydii bearing the antigen OC1 was obtained from the National Collection of Type Cultures (NCTC accession No. 9731) and tested against standard reference typing sera to confirm its typing. More specifically, the Shigella boydii was removed from the lyophile, grown on blood agar, and tested by conventional biochemical (API) and agglutination tests with appropriate antisera to confirm it identity and purity. The cells were then transferred to DMEM, grown, and harvested for use as a source of antigen. The organisms were washed in phenol saline by repeated centrifugation and were resuspended in phenol saline. 3. Animal Immunisatio si Balb/c mice were injected with the prepared antigen. They were given one intraperitoneal injection per week for three weeks (0.05 ml 80% T vaccine) followed by six intravenous injections every other week of LDlO of boiled killed Shigella boydii 0C1 vaccine prepared as above for a total of eleven 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 of 80% T vaccine intravenously, three days prior to splenectomy. C. Cell Fusion The selected donor mouse was killed and surface sterilised by immersion in 70% ethyl alcohol. The spleen was then removed and immersed in approximately 2.5 ml of 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 of 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 a tissue culture medium DMEM containing 3% FCS.

The spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 6.00 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. Over the period of one minute, 1 ml of a 50% w/v solution of polyethylene glycol 1500 (PEG) in saline

Hepes, pH of approximately 7.5, is added, and the mixture gently stirred for approximately 1.5 minutes. There were then slowly added 10 ml of serum-free tissue culture medium DMEM, followed by the addition of 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 multiwell tissue culture plates. Each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin, and thymidine) and a feeder 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% C0₂-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 Shigella boydii OC1 antigen was developed in at least one well. D. Cloning

From those wells which yielded antibody against the Shigella boydii OC1 antigen, cells were removed and cloned using the standard agar method and by limiting dilution.

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_, 9.5% RH. Viable separate colonies were picked off the agar surface and placed into 60 wells of a 96-well microtitre tray in 18% FCS-DMEM. After a further period of growth, the supernatants were assayed for specific antibody by the standard enzyme immunosorbent assay.

In limiting dilution, dilutions of cell suspensions in 18% FC-DMEM + Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtitre plate. The plates were incubated for 7-14 days at 37°C, 95% RH, 7-9% CO., until semi-confluent. The supernatants were then 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 Shigella boydii NCTC 9731 prepared as in the immunisation, and for specificity in a tes battery of Shigella species and related genera bearing different antigens. Specifically, a grid of microtiter plates- containing a representative selection of O-serotype organisms, i.e. Pseudomonas, Klebsiella, Serratia and Enterobacter, was prepared, boiled, and utilised as a template to define the specificity of the parent

O-specifi.c group. The EIA immunoassay noted above was used.

F. Antibody Production

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

7 days, and injected intraperitoneally with 10 cells of the monoclonal antibody-producing 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.

The fluid was titrated, as noted above, to establish presence and level of antibody, and purified. Purification is accomplished using the Protein A-Sepharose method, in which about 10 ml of the ascites fluid are 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 (0.1 M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose which had previously been equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1 M sodium citrate, pH 3.5) into sufficient 1M Tris buffer, pH 9.0, to raise the pH immediately to about 7.5. The eluate was dialysed in 2 x 1000 ml PBS, pH 7.4 at +4°C, and stored at -20°C. G. Enzyme-Monoclonal Linkage

The monoclonal antibody specific against Shigella boydii 0C1 antigen, prepared and screened as described above, was then bound to an appropriate enzyme (in this case, a 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. After dialysis, the volume was made up to 2.5 ml with PBS and 25 ul of a 20% solution of glutaraldehyde in PBS was 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 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. 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 assay method comprises coating the wells of a standard polyvinyl chloride microtiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenol phosphate.

In this case, the monoclonal antibody was found to be specific for the OC1 antigen of Shigella boydii. The monoclonal antibody was also tested and shown to be of the Class IgG3.

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 11

The procedure of Example 1 was followed in each of 10 cases, with differences outlined below, to prepare monoclonal antibody conjugates for various antigens of the genus Shigella.

In Examples 2 to 7, Shigellae boydii bearing the respective antigens OC4, OC6, OC7, OC10 and OCll (twice) were used; in Example 8, Shigella flexneri bearing the OBI antigen; in Examples 9 and 10, Shigellae dysenteriae bearing the OAl and OA10 antigens; and, in Example 11, Shigella sonnei bearing the OD 1 antigen. These were all obtained from NCTC where the respective accession numbers are 9330, 9332, 9333, 9357, 9321, title 3, 4837, 9351 and 9774.

In the animal immunisation step for Examples 2, 4, 6, 7, 8, 10 and 11, Balb/c mice were vaccinated intraperitoneally with the prepared antigen (0.05 ml of 80% T vaccine) followed by an intravenous boost after six weeks. When the mice showed a suitably elevated titre to the immunising antigen, they were used as a source of spleen cells for fusion.

In the animal immunisation step for Example 3, the mice were immunised with a dose of 0.05 ml of 80% T vaccine given in one intraperitoneal injection per week for three weeks followed by one intravenous injection per month, for three months, of boiled, killed Shigella boydii OC6 prepared as in Example 1. In the animal immunisation step for Example 5, the mice were immunised with a dose of 0.5 ml of 80% T vaccine given in one intraperitoneal injection per week for three weeks, followed by intravenous injection per week for two weeks, of boiled and killed S_. boydii prepared as in Example 1, for a total of five weeks.

In the animal immunisation step for Example 9, the mice were immunised with a dose of 0.05 ml of 80% T vaccine given in one intraperitoneal injection per week for three weeks, followed by an intravenous injection per week for three weeks, and then four intravenous injections every other week, of boiled, killed Shigella dysenteriae, for a total of 14 weeks.

In the antigen preparation step for Examples 2, 3 and 5 to 10, the organisms were boiled as well as washed, in phenol saline. In Examples 4 and 11, the organisms were boiled and washed in saline. In Example 11, resuspension was in phenol saline.

In the cell fusion step for Examples 2, 8 and 10, 6

7 7 x 10 myeloma cells were used; 7 x 10 in Example 5; and

8 8 1 x 10 in Examples 6 and 7. 1.5 x 10 spleen cells were g used in Example 2, and 1.2 x 10 in Examples 6 and 7.

In the cloning step for Examples 2 and 6 to 10, the agar method alone was used; in Examples 3 and 5, the limiting dilution and agar methods; and, in Examples 4 and 11, the limiting dilution method (twice). In the monoclonal selection step, Shigella and E. coli were invariably used, Pseudomonas in all except Examples 3 and 7, Klebsiella in all except Examples 2, 3, 7 and 8, Salmonella in all except Example 4, and Serratia in Example 2 only.

In the antibody production step for Examples 3, 4 and 11, cells of the monoclonal antibody-producing cell line were grown in bath tissue culture. 10% FCS-DMEM was used to support growth in mid-log phase, to 1 litre volume, and then the culture was allowed to overgrow, to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 min. The cells were discarded and the antibody-rich supernatant collected. The antibody purification step for Examples 3 and 4 involved a supernatant on SP-Sephadex method. To 1 litre of culture supernatant was added 1 litre of 0.05M sodium acetate buffer, pH 4.5, and 40 ml of SP-Sephadex, previously equilibrated in 0.1M 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 0.1M acetate buffer, pH 5.0, and eluted with 60 ml of the same buffer plus 1M 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 min, and then the precipitate was harvested by centrifugation at 10,000 g for 10 min. The precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) . The dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 min and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer. The monoclonal antibody was eluted with phosphate/EDTA buffer.

The antibody purification step for Examples 6, 8, 9 and 10 was accomplished using the ammonium sulphate precipitation/DEAE-cellulose method. Ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 min. The ascites was then stirred at +4°C and an equal volume of cold, saturated ammonium sulphate added slowly. The mixture was stirred for a further 30 min after the addition was complete. The precipitate was harvested by centrifugation at 10,000 g for 10 min. The precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, +- 0.02% sodium azide) . The. solution was dialysed vs 2 x 1000 ml of the same buffer at +4°C. The dialysed, redissolved precipitate was centrifuged at 30,000 for 10 min and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer. The monoclonal antibody was eluted with phosphate/EDTA buffer.

The antibody purification step for Example 11 involved the supernatant on Protein A-Sepharose method. To 1 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 the -antibody conjugation step for Examples 3, 5, 6, 8 and 11, the benzoquinone method was used. 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25M sodium phosphate buffer, pH 6.0, at +4°C. Para-benzoquinone, 18 mg, was dissolved in warm AR ethanol, 0.6 ml, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. After this time, unreacted benzoquinone and reaction by-products were removed and the buffer exchanged, by gel filtration on a Pharmacia PD-10 (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 8 mg- of benzoquinone-activated alkaline phosphatase, 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. After this time, sufficient IM lysine was 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 of PBS + 0.02% sodium azide at +4°C. An equal volume of glycerol was added. The conjugate was sterile- filtered through a 0.22 μm membrane filter into a sterile amber vial and stored at +4°C.

Conjugate testing showed the appropriate specificity in each case. The monoclonal antibodies were also tested and shown to be of the classes IgG2a (Examples 2, 5 and 6), IgG3 (Examples 3, 9, 10 and 11), IgGl (Examples 4 and 7) and IgM (Example 8) . Example 12

The same procedure as in Example 1 may be utilized in preparing a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Shigella, but using another Shigella obtained from the National Collection of Type Cultures.

Tests using the present invention are superior to the existing tests based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expense; and (v) improved therapy based upon early, precise diagnosis.

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 Shigella.

2. The antibody of Claim 1 specific to the 0A1 antigen of Shigella dysenteriae.

3. The antibody of Claim 1 specific to the 0A2 antigen of Shigella dysenteriae.

4. The antibody of Claim 1 specific to the OA3 antigen of Shigella dysenteriae.

5. The antibody of Claim 1 specific to the 0A4 antigen of Shigella dysenteriae.

6. The antibody of Claim 1 specific to the OA5 antigen of Shigella dysenteriae.

7. The antibody of Claim 1 specific to the 0A6 antigen of Shigella dysenteriae.

8. The antibody of Claim 1 specific to the 0A7 antigen of Shigella dysenteriae.

9. The antibody of Claim 1 specific to the OA8 antigen of Shigella dysenteriae.

10. The antibody of Claim 1 specific to the OA9 antigen of Shigella dysenteriae.

11. The antibody of Claim 1 specific to the OA10 antigen of Shigella dysenteriae.

12. The antibody of Claim 1 specific to the OBI antigen of Shigella flexneri.

13. The antibody of Claim 1 specific to the OB2 antigen of Shigella flexneri.

14. The antibody of Claim 1 specific to the OB3 antigen of Shigella flexneri.

15. The antibody of Claim 1 specific to the 0B4 antigen of Shigella flexneri.

16. The antibody of Claim 1^' specific to the OB5 antigen of Shigella flexneri.

17. The antibody of Claim 1 specific to the OB6 antigen of Shigella flexneri.

18. The antibody of Claim 1 specific to the OC1 antigen of Shigella boydii.

19. The antibody of Claim 1 specific to the OC2 antigen of Shigella boydii.

20. The antibody of Claim 1 specific to the OC3 antigen of Shigella boydii.

21. The antibody of Claim 1 specific to the OC4 antigen of Shigella boydii.

22. The antibody of Claim 1 specific to the OC5 antigen of Shigella boydii.

23. The antibody of Claim 1 specific ^* to the OC6 antigen of Shigella boydii.

24. The antibody of Claim 1 specific to the 0C7 antigen of Shigella boydii.

25. The antibody of Claim 1 specific to the 0C8 antigen of Shigella boydii.

26. The antibody of Claim 1 specific to the 0C9 antigen of Shigella boydii.

27. The antibody of Claim 1 specific to the OC10 antigen of Shigella boydii.

28. The antibody of Claim 1 specific to the 0C11 antigen of Shigella boydii.

29. The antibody of Claim 1 specific to the OC12 antigen of Shigella boydii.

30. The antibody of Claim 1 specific to the OC13 antigen of Shigella boydii.

31. The antibody of Claim 1 specific to the 0C14 antigen of Shigella boydii.

32. The antibody of Claim 1 specific to the OC15 antigen of Shigella boydii.

33. The antibody of Claim 1 specific to the OD1 antigen of Shigella sonnei.

34. The antibody of Claim 1 specific to the invasiveness 1 antigen of Shigella.

35. The antibody of Claim 1 specific to the invasiveness 2 antigen of Shigella.

36. The antibody of Claim 1 specific for the enterotoxin 1 of Shigella.

37. The antibody of Claim 1 specific for the enterotoxin 2 of Shigella.

38. The antibody of Claim 1 specific for attachment antigen 1 of Shigella.

39. The antibody of Claim 1 specific for attachment antigen 2 of Shigella.

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

41. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 1-40 and an appropriate label.

42. The labeled monoclonal antibody of Claim 41, wherein said label is a member of the group selected from a radioactive isotope, enzyme, fluorescent compound, bioluminescent compound, chemiluminescent compound, or ferro¬ magnetic atom, or particle.

43. The labeled monoclonal antibody of Claim 42, wherein said label is an enzyme capable of conjugating with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure.

44. The labeled monoclonal antibody of Claim 43, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase.

45. The labeled monoclonal antibody of Claim 42, 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.

46. The labeled monoclonal antibody of Claim 45, wherein said fluorescent compound or probe is fluorescein.

47. The labeled monoclonal antibody of

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

48. The labeled monoclonal antibody of Claim 47, wherein such chemiluminescent compound is luminol or a luminol derivative.

49. The labeled monoclonal antibody of Claim 42, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay.

50. The labeled monoclonal antibody of Claim 49, wherein such bioluminescent compound is luciferase or a luciferase derivative.

51. A process for diagnosing for the pre¬ sence of an antigen of Shigella in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 41 in an immunoassay procedure appropri¬ ate for said label.

52. The process of Claim 51, 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.

53. The process of Claim 52, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure.

54. The process of Claim 53, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase.

55. The process of Claim 52, 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.

56. The process of Claim 55, wherein said fluorescent compound or probe is fluorescein.

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

58. The process of Claim 57, wherein, said chemiluminescent compound is luminol or a luminol derivative.

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

60. The process of Claim 59, wherein said bioluminescent compound is luciferase or a lucif- erase derivative.

61. A therapeutic composition comprising one or more of the monoclonal antibodies of Claims 1-40 and a pharmaceutically acceptable carrier or diluent.

62. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 41 and a pharmaceutically acceptable carrier.or diluent.

63. A method of treating shigellosis com¬ prising administering an effective amount of a monoclonal antibody of Claims 1-40.

64. A kit for diagnosing for the presence of an antigen or species of Shigella in a diagnos¬ tic specimen comprising at least one monoclonal antibody of Claims 1-40.

65. The kit of Claim 64, wherein said at least one antibody is labeled.

66. The kit of Claim 65, wherein said at least one monoclonal antibody is labeled with a fluorescent compound.

67. The kit as in Claim 65, wherein said at least one monoclonal antibody is labeled with an enzyme.

68. The kit as in Claim 65, 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.

69. The kit of Claims 65, 66, 67, and 68 additionally containing at least one known Shigella antigen as a control.

70. The kit of Claims 65, 66, 67, 68, and 69 containing each known antigen of Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei.

71. The kit of Claims 65, 66, 67, 68, and 69 containing the OA1, OA2, OA3, OA4, OA5, OA6, OA7, OA8, OA9, or OA10 antigens of Shigella dysenteriae.

72. The kit of Claims 65, 66, 67, 68, and 69 containing the OBI, OB2, OB3, OB4, OB5, or OB6 antigens of Shigella flexneri.

73. The kit of Claims 65, 66, 67, 68, and 69 containing the OC1, OC2, OC3, OC4, OC5, OC6, OC7, OC8, OC9, OC10, OC11, OC12, OC13, OC14, or OC15 antigens of Shigella boydii.

74. The kit of Claims 65, 66, 67, 68, and 69 containing the ODl antigens of Shigella sonnei.

75. The kit of Claims 65, 66, 67, 68, and 69 containing invasiveness antigens 1 and 2 of Shigella.

76. The kit of Claims 65, 66, 67, 68, and 69 containing enterotoxin 1 and 2 of Shigella.

77. The kit of Claims 65, 66, 67, 68, and 69 containing attachment antigens 1 and 2 of Shigella.

78. A kit for diagnosing for the. presence of an antigen or species of Shigella in a diagnos¬ tic specimen comprising at least one monoclonal antibody of Claims 1-40 and a control.

79. The kit of Claim 78, wherein said at least one antigen is labeled and said control is at least one known antigen of Shigella.

80. A kit for diagnosing for the presence of a gram-negative bacterial infection comprising at least one monoclonal antibody of Claims 1-40.

81. The kit of Claim 80, wherein said at least one monoclonal antibody is labeled.