WO1987006468A1 - Monoclonal antibodies and their use - Google Patents

Abstract

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

Description

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MONOCLONAL ANTIBODIES AND THEIR USE FIELD OF THE INVENTION

This invention relates to monoclonal antibodies and their use. BACKGROUND OF THE INVENTION

Organisms (gram-negative) of the genus Mobiluncus are inhabitants of the vaginal tract. They are implicated in vaginosis syndrome.

Species of the genus are Mobiluncus mulieris and Mobiluncus curtisii. Among the latter, there are subspecies holmesii and curtisii.

Existing methods of detection of Mobiluncus with high accuracy may be 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 Mobiluncus antigens and/or organisms.

Briefly stated, the present invention comprises monoclonal antibodies specific for an antigen of Mobiluncus; in particular, the antigens of Mobiluncus mulieris and Mobiluncus curtisii (inc. subsp. holmesii and curtisii) , as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Mobiluncus.

The invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Mobiluncus antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens or to a particular species thereof and having linked thereto an appropriate label. The label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom, or particle, or any other label.

The invention further comprises the process for diagnosing the presence of Mobiluncus antigens or organisms in a specimen comprising contacting said specimen with the labelled monoclonal antibody in an appropriate im unoassay procedure.

Additionally, the invention is also directed to a therapeutic composition comprising a monoclonal antibody for an antigen of Mobiluncus and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of Mobiluncus.

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 Mobiluncus 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, 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 chosen Mobiluncus antigen over a suitable period, e.g. 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 Mobiluncus 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 give a positive response to the presence of the particular Mobiluncus 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 Mobiluncus antigen. The monoclonal antibody selected, which is specific for the particular Mobiluncus 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 a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like. The present invention will be described with reference to the use of an enzyme labelled monoclonal antibody. Some of the enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, urease, and the like.

Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.

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 due to their low cost, reagent stability, safety, sensitivity, and ease of procedure. One example is 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 immunoglobulin 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 non-labelled 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 heterogenous 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 nano-mole 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 Mobiluncus 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 Mobiluncus infections and they are used in amounts effective to cure; an amount which will vary widely dependent 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 Mobiluncus in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Mobiluncus alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Mobiluncus and/or other bacteria. In the past there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens such as urine with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis. For example, a kit can be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis.

Additionally, conjugated or labelled monoclonal antibodies for antigens and/or species of Mobiluncus 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 Mobiluncus 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.

In addition to being sold individually, the kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Mobiluncus.

One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Mobiluncus antigen or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Mobiluncus antigen and/or a cross-reactive labelled monoclonal antibody which can detect the presence of any of the Mobiluncus organisms in a particular sample. Specific antigens to be detected in this kit include the antigen of Mobiluncus mulieris or Mobiluncus curtisii (or a subspecies thereof) .

Monoclonal diagnostics which detect the presence of Mobiluncus 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 Mobiluncus antigen. More particularly, products of 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 contamina¬ tion.

The invention will be further illustrated in connection with the following Examples which are set forth for the purposes of illustration .only and not by • way of limitation.

The monoclonal antibody of the present invention was prepared generally according to the method of Kohler and Milstein, Eur. J. Immunol. 6_ (1975) 292. 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

CFA = complete Freunds adjuvant

IFA = incomplete Freunds adjuvant im = intramuscular iv = intravenous Example 1

A. Antigen Preparation

Mobiluncus curtisii subsp. curtisii antigen was obtained from the National Collection of Type Cultures (NCTC 11656) . It may be tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . More specifically, the antigen may be removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity. The blood agar is scraped off into formol saline, as a source of antigen. The organisms were washed in formol saline by repeated centrifugation and were finally resuspended in formol saline.

B. Animal Immunisation Balb/c mice were injected with the prepared antigen. They were given two im injections + CFA, with an interval of 2 weeks between the intramuscular injections. The mice were rested for 6 weeks before being given a further iv injection + PBS. 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 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.

Using the myeloma cell line NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England, myeloma cells were obtained 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 was 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.

1.14 x 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 a period of one minute, 1 ml of a 50% w/v solution of polyethylene glycol 1500 (PEG) in saline Hepes, pH 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 multi-well 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 5x10 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 Mobiluncus antigen was developed in at least one well.

D. Cloning ' From those wells which yielded antibody against the

Mobiluncus antigen, cells were removed and cloned using the limiting dilution method.

Dilutions of cell suspensions in 18% FCS-DMEM +

Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtiter 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. E. Monoclonal Selection

The monoclonal antibodies from the clones were screened by the standard techniques for binding to the Mobiluncus antigen prepared as in the immunisation, and for specificity in a test battery of Mobiluncus species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms was prepared, boiled, and utilised as a template to define the specificity of the parent O-specific group. The EIA immunoassay noted above was used.

Two monoclonal antibodies were selected in this case, respectively of subclasses IgG2b and IgGl. Each cross-reacts with NCTC 11646 and NCTC 11657 (M. curtisii subsp. holmes) .

F. Bulk Antibody Production

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. G. Antibody Purification

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 0.1M 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 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. H. Enzyme-Monoclonal Linkage

The monoclonal antibody specific against the Mobiluncus antigen, prepared and screened as described above, was then bound to an appropriate enzyme (in this case, a highly purified alkaline phosphatase) .

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.

I. 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 antigen of Mobiluncus. The monoclonal antibody was also tested and shown to be of the immunoglobulin Class.

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. Example 2

The same procedure as in Example 1 may be utilised to prepare a monoclonal antibody from M. curtisii subsp. holmes (NCTC 11657) , with the exceptions that the second im injection used IFA rather than CFA, the rest period p between im and iv injections was 7 weeks, 2.5 x 10

7 spleen cells were mixed with 2.5 x 10 myeloma cells, and

240 wells were used. The subclass is IgGl. The same cross-reactivity is observed as in Example 1. Example 3

The same procedure as in Example 2 may be utilised to prepare a monoclonal antibody from M. mulieris (NCTC 11658) , with the exceptions that the myeloma cells were

^•7 Sp20 and were mixed with 5 x 10 spleen cells.

Antibodies of subclasses IgGl and IgG2a were obtained, specific to the NCTC 11658 antigen. Example 4

The same procedure as in Example 1, 2 or 3 may be utilised to prepare a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Mobiluncus, but using another Mobiluncus antigen 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

1. A monoclonal antibody specific for an antigen or species of Mobiluncus.

2. A monoclonal antibody according to claim 1, which is specific to the antigen or antigens of Mobiluncus mulieris or Mobiluncus curtisii (subsp. holmesii or curtisii) , or which is broadly cross-reactive with an antigen of all species of the genus Mobiluncus.

3. A monoclonal antibody according to claim 1 or claim 2, which is labelled.

4. A monoclonal antibody according to claim 3, wherein the label is a radioactive isotope, enzyme, fluorescent compound, bio-luminescent compound, chemi-luminescent compound, or ferromagnetic atom or particle.

5. A monoclonal antibody according to claim 4, wherein the label is an enzyme capable of being used in an enzyme-linked immunoassay procedure, a fluorescent compound or probe capable of being used in an immuno-fluor scent, fluorescent, enzyme-fluorescent, fluorescence-polarisation or photon-counting immunoassay procedure, a chemi-luminescent compound capable of being used in a luminescent or enzyme-linked immunoassay procedure, or a bio-luminescent compound capable of being used in a bio-luminescent immunoassay procedure.

6. A monoclonal antibody according to claim 5, wherein the label is an enzyme selected from alkaline phosphatase, glucose oxidase, galactosidase and peroxidase, fluorescein, a chemi-luminescent compound selected from luminol and luminol derivatives, or a bio-luminescent compound selected from luciferase and luciferase derivatives.

7. A monoclonal antibody according to any preceding claim, for use in treating Mobiluncus infections.

8. A process for diagnosing for the presence of an antigen of Mobiluncus in a specimen, which comprises contacting the specimen with a monoclonal antibody according according to any of claims 4 to 6 in an immunoassay procedure appropriate to the label.

9. A therapeutic composition which comprises a monoclonal antibody according to any of claims 1 to 6 and a pharmaceutically-acσeptable carrier or diluent.

10. A kit for diagnosing for the presence of a gram-negative bacterial infection, which comprises a monoclonal antibody according to any of claims 1 to 6 and, as a control, a known Mobiluncus antigen.