GB2309459A - Antibodies to fungal antigens - Google Patents

Description

2309459 ANTIBODIES TO FUNGAL ANTIGENS The invention relates to the raising

of antibodies against fungal antigens.

Mould fungi are prevalent in nature. Some of these fungi are useful to man in that they produce antibiotics. others are saprophytic and can infect food rendering it unfit for consumption due to the production of mycotoxins. There are other fungi which can infect humans or animals directly and which have pathological effect. For example, chickens fed on chicken feed infected with one or more Asipercrillus sp., Microsporum sp., Mucor sp. and Penicillium sp. may exhibit a variety of disease states, for example infertility or tumours on internal organs, such as the liver, kidney or is spleen. The mouldy feed may even cause the death of birds.

A known method of detecting the presence of contaminating fungi in foodstuffs consists of taking a sample of the food and then culturing it in a suitable growth medium. Any fungi present in the sample can multiply in the growth medium. The amount of fungal growth in culture after a given time indicates the amount of the fungus in the sample at the time of sampling. However, a disadvantage of this method is that only viable fungi or fungal spores in the sample will be detected. If the sample contains non-viable fungi or dormant spores then the test can give a negative result. Even where there is a negative test result there may still be mycotoxins left in the food from a period of fungal infection.

The test method employing the culturing of samples suffers from the particular disadvantage that it takes a number of days for the test cultures to grow. A test by this method cannot provide results rapidly. Therefore if an animal is infected then the infection would continue whilst the testing was concluded. Although no symptoms of infection might be seen in the animal it could have reached a stage of infection where treatment was needed in order to prevent the onset of disease symptoms or action needed to isolate the animal from others to prevent the spread of infection. A rapid test for fungal infection would allow the earlier treatment of the animal or its isolation from others.

Yet another method of detecting fungal infection is to examine an infected sample under the microscope. A disadvantage of this method is that it is laborious and it is susceptible to error. In practice this test is not particularly accurate and there can be a wide variation in the results obtained by this method.

Where there are fungus specific chemical compounds in a sample then these can be detected and quantified. For example, the aflatoxins produced by species of the genus Asipercrillus can be detected by chromatographic techniques.

Heat stable mould enzymes can be assayed. Also, fungi have is cell walls made of chitin and a chemical constituent of chitin is glucosamine. After breaking down the chitin with enzymes or chemically, the amount of glucosamine released can be determined colorimetrically. A disadvantage of detecting particular chemical compounds or enzymes in samples by biochemical or physicochemical techniques is that the samples usually need some kind of preparation and this is laborious and time consuming. Also, a reasonable quantity of sample material is needed in order to provide a sufficient quantity of chemical compound for detection. Sometimes the problem arises that the chemical compound being determined is not truly fungus specific but is sometimes present in a foodstuff under test as a naturally occurring component of that foodstuff.

EP-0325004 (De Staat Der Nederlanden) seeks to provide a solution to the problem of detecting moulds in foodstuffs and in animal body fluids. The object of this invention is to provide an immunoassay for Penicillium and Asipergillus which does not generate false positive results. The invention lies in raising antibodies against extracelluar poly saccharides (EPS) isolated from cultures of Asipergillus or Penicillium. The EPS has powerful antigenic properties and is virtually genus specific. The antibodies are then used in a sandwich enzyme-linked immunoadsorbent assay (ELISA) wherein the antibodies are immobilised in the wells of a microtiter plate and then presented with test samples. Antigens recognised by the antibodies will be retained by the antibodies, any unrecognised and therefore unbound antigens can be washed away. Further antibody linked to a marker enzyme is then added to the wells of the microtiter plate. This binds to any available antigen which is itself bound to the immobilised antibodies. The addition of an appropriate enzyme substrate gives a coloured product which can be observed and measured. In order to show where a positive reaction is false, an identical test is carried out on each sample but in the presence of a concentration of a synthetic is antigen which is selected to give a fifty percent inhibition of any response. The synthetic antigen acts by out-competing any antigen in the test sample for the immobilised antibody recognition sites. The synthetic antigen is constructed so that it contains just one epitope; the enzymelinked antibodies added later will not be able to recognise the synthetic antigen when is bound by that single epitope to the immobilised antibodies. No recognisable epitope will be presented by the synthetic antigen if it is already antibodybound. A reduction in enzyme product in the microtiter plate wells will be seen when the synthetic antigen out competes any native antigen in the test sample. If no reduction in enzyme product is seen with the synthetic antigen then any positive result on that sample could not be relied on.

The antibodies of EP 0325004 can be against genus specific EPS antigens from a wide variety of moulds including Aspergillus. , Penicillium, Geotrichum, Fusarium, Cladosporium, Mucor and Rhizopus.

US 4835100 (Neogen) concerns the raising of monoclonal antibodies against aflatoxins produced by Aspergillus. The monoclonal antibodies are prepared from material derived from the subcutaneous immunisation of mice with purified af latoxins conjugated to a carrier. The monoclonal antibodies are used to provide an immunochemical test for detecting the presence of aflatoxins in a sample.

US 4772551 (Neogen) concerns the production of monoclonal antibodies to a trichothecene mycotoxin. These toxins are produced mainly by species of Fusarium. The monoclonal antibodies are raised from material derived from the subcutaneous immunization of mice with isolated toxins coupled to carriers. The monoclonal antibodies produced are used to detect the aflatoxins by way of an ELISA for example.

The abstract of Preston et al in Chemical Abstracts 73: (1970) concerns rabbit antisera raised against extracelluar polysaccharides of Penicillium. The antibodies are raised by injecting rabbits with whole cells of Penicillium.

An improved immunochemical assay for fungal antigens would provide a test method which was rapid and required only a small quantity of sample. However, there are no commercially available antisera or antibodies against fungal antigens. It is widely recognised in the f ield that it is extremely difficult to generate antibodies of a reasonable titer against a single isolated fungus (Environmental Carcinogens and Selected Methods of Analyses Vol. 5 Some Mycotoxins Eds:- Egan H., Stalof L., Castegnaro M. et al; International Agency for Research on Cancer, Lyon (1982)).

The inventor has determined that it is not possible to raise antibodies in chickens simply by feeding the chickens on feed contaminated with a single fungus.

The invention provides antibodies immunoreactive against one or more. e.g two or more, of Fusarium, Aspercrillus, Mucor and Micros-porum such that an immunoreaction takes place when the antibodies are challenged with one or more of their respective eliciting antigens.

Preferably, the antibodies do not exhibit any immunoreactivity against Penicillium. If there is any immuno- reactivity against Penicillium then this would be of a low titer and possibly non-specific.

-5 Preferably, the immunoreaction is capable of being observed as an immunoprecipitate. The immunodetection may however not always be by observation of an immunoprecipitate but may be by another immunochemical method such as radioimmunoassay or ELISA for example.

The antibodies are preferably polyclonal.

The polyclonal antibodies may be monospecific against the said one or more eliciting antigens. The monospecificity may be obtained by the immunoaffinity purification of the polyclonal antibodies.

The antibodies may be monoclonal. In preparing monoclonal antibodies a known method of antibody-producing hybridoma formation may be used.

The antibodies may be provided in the form of an is antiserum.

The antibodies may be conjugated to a protein, enzyme, radioisotope or a chemical compound thereby enabling the specific detection of the antibodies by some standard method of detection known to the worker skilled in this field.

The invention includes the use in an immunoassay, immunodetection or immunolocalisation technique against selected fungal antigens of the antibodies or antisera herein defined.

The invention includes a test kit for detecting selected fungal antigens comprising any of the antibodies herein defined.

Suitable antibodies may be raised against a fungal antigen by exposing the immune system of an organism to one or more eliciting fungal antigens in the presence of at least one substantially non-immunogenic fungal antigen of Penicillium, and then extracting the immune serum from the organism.

The reference to a fungal antigen includes a single antigenic determinant from a fungal source or a plurality of antigens from fungal sources each having a multiplicity of antigenic determinants.

The substantially non-immunogenic fungal antigen of Penicillium is nonimmunogenic to the extent that either no antibodies are raised against it or if any antibodies are indeed raised to it then they are of poor titer and possibly also non-specific.

The Penicillium may act as an adjuvant.

The preferred organism is a chicken. However, other birds may be used. Also, other organisms such as mammals may be used.

The eliciting antigens are preferably administered orally to the organism or they may be administered directly into the digestive tract. Other methods of immunisation may be used such as intramuscular, subcutaneous or intravenous injection.

The eliciting antigens are preferably mixed with the food is of the organism. The presence of the fungal antigens in the food may arise naturally through infection or purposefully by adding cultures of selected fungi to the food.

Preferably the eliciting antigens consist of fungal mycelia. The fungal mycelia may be fractionated into defined 20 cellular or extracellular components.

The mycelia may first be killed by heat or radiation treatment before administration to the organism.

The eliciting antigens may be extracts of mycelia. The extracts may be partially purified using standard biochemical 25 preparation techniques.

The eliciting antigens may be extracted using an organic solvent. The extraction of the antigens may take place either using whole mycelia as the starting material or by using partially purified extracts of these mycelia.

Preferably, the organic solvent is ethanol.

The mycotoxins will usually be present in infected foodstuffs ingested by the organism or they will usually be released by fungi which have already infected the organism.

The antibodies may be used in a method of detecting the presence of a particular fungal antigen in a sample comprising challenging a first aliquot of the sample with first is antibodies immunoreactive against a combination of fungal antigens not including said particular antigen and challenging a second aliquot of the sample with second antibodies immunoreactive against the particular antigen and against said combination of fungal antigens, wherein an immunoreaction with the second antibodies and no immunoreaction with the f irst antibodies indicates that the sample contains the said particular fungal antigen but not any of the said combination of antigens.

The particular fungal antigen may be produced by Fusarium. Alternatively, particular fungal antigens may be produced by one of Aspercrillus, Mucor or Microsporum. The particular fungal antigen may be produced by a genus of fungus known to the worker skilled in the art.

The said combination of fungal antigens is preferably one or more of Aspergillus, Mucor and Microsporum. Other combinations of mould fungi known to the worker skilled in the art are possible.

Preferably, the immunoreaction is an immunoprecipitation which can be observed by the naked eye or under the light microscope.

The antibodies may be labelled with a marker and the immunoreaction may be detected by reference to the said marker. The marker may be a radioisotope, an enzyme, a protein, a chemical compound such as biotin, streptavidin or fluorescein, or a combination of markers.

The immunoreaction may be detected by way of an enzymelinked immunoadsorbent assay (ELISA).

The invention will now be described by way of the following examples.

Example 1

Generation of Polyclonal Antibodies Type A Polyclonal Antibodies Two thousand eighteen-week old chickens were fed on mouldy feed naturally contaminated with Aspergillus, Microsporu, Mucor and Penicillium. The feed was Chinese soya which was 45.29k by weight protein, 4.8% by weight fat and had a urease enzyme activity of 2.18 mg Urea N11 (the usual value for urease activity in Chinese soya is 0.30 mg Urea N11 or less). The chickens were fed on the feed for three days.

After feeding, two in three chickens died, and one in six chickens were infected and developed mycosis. The infected chickens exhibiting mycosis were used for antigen preparation. The remaining one in six chickens became infected but recovered from the infection. These chickens developed antisera (Type A Polyclonal Antibodies) against the feed contaminating fungi.

Type B Polyclonal Antibodies One thousand eighteen-week old chickens were fed on mouldy feed naturally contaminated with Asipergillus, Microsporum, Mucor, Penicillium and Fusarium as described above in relation to the Type A Polyclonal Antibodies. Eight out of ten of the chickens died. One in ten chickens developed mycosis and liver tumours. The remaining one in ten chickens became infected and survived having developed antibodies against the infecting fungi (Type B Polyclonal Antibodies).

The type A and Type B antibodies were obtained from the chickens by bleeding them. The blood samples were kept on ice and then centrifuged for fifteen minutes at 200Oxg. The supernatant was taken and then divided into 100 gl aliquots. These aliquots were frozen until needed.

Examiple 2 Antigen PreDaration and Mycotoxin Extraction (not an examiple of the invention) In order to determine the immunoreactivity of the antibodies, quantities of the selected antigens are needed.

To prepare these, one gram of contaminated feed was ground in a sterile pestle and mortar and then extracted with 10 ml of sterile distilled water. The feed was soya beans and in the case of antigen A the feed was contaminated with Aspergillus, Microsporum, Mucor and Penicillium. In the case of antigen B the feed was contaminated with Asipercillus, Microsporum.

Mucor, Penicillium and Fusarium.

The aqueous extract so prepared was then centrifuged.

is A loopful of a resulting supernatant was cultured on fungal selective media being:

(1) Sabraud-glucose agar (2) Litman's Oxagoll agar (3) Blood Agar.

Fungal growth developed within five days. After seven days giant colonies of fungi were found.

A wet mount of selected colonies of each fungus from the fungal selective media (1) and (2) were examined under the light microscope in order to identify the fungi and their spores. The results of the analyses of the fungal colonies isolated from the contaminated feed is set out in Tables 1 and 2 below.

TABLE 1

Molphological Characteristics and Scrological RespQnses of Various Fungi to I.Xpe A Polyclonal Antiserum Fungal Genus Morphological Characteristics Serological Response Colony Spores Type A Agglutination Test As=gillus Black 42>. e> Pigmented black Positive spores Micrn-,porum Yellow Positive While, Creamy Mucor Brown, Fluffy 00 0 Positive Penicilflurn Green Green-blue Negative Pigmented spores TABLE 2

Molphological Characteristics and SernIngical Responses of Various Fungi to lype B Polyclonal Antigerum Fungal Genus Morphological Characteristics Serological Response Colony Spores Type B Agglutination Test Black Black pigmented Positive Orange Positive Microsporum Yellow Positive White creamy Mucor Brown fluffy Positive Penicillium Green Green-blue Negative pigniented For detailed morphological features of these fungi appearing in Tables 1 and 2, see:

(a) Koneman, E, W & Roberts, G. D. in 'Tractical Laboratory Mycology" 13rd Edition, Williams & Wilkins (1985) pp 86-92, 118-119, i106 & 201; and (b) Koneman, E.W., Alien, S.D. Dowell, V.R. Jr. & Sommers, H M, in "Color Atlas & Text Book of Diagnostic Microbiology% 3rd Edition, JB Lippincot Company, (1983) pp 523, 533 & 545.

The morphological identification of the fungal colonies was correlated with an agglutination test using antibodies A 10 and B. The agglutination tests are described in Example 4.

When blood agar medium was used, no bacterial growth was observed thereby suggesting the absence of coliforms.

Non-contaminated feed was extracted in the same way as contaminated feed and samples of this were cultured on the is fungal selective media. No fungal or bacterial growth was found on these control culture plates.

The contaminated feed includes soya beans, Chinese soya, rice, corn, peanuts (shelled or unshelled) or hazelnuts.

Example 3

Antigens obtained from Biopses of Infected Chickens (not an example of the invention) Another source of fungal antigens to use in the characterisation of the antibodies is from the infected chickens themselves.

Small pieces of infected liver, kidney or spleen were cultured on fungal selective media. Where the chickens had been fed on contaminated feed, the range of fungal species found contaminating the feed were also found to be present in various internal organs of the chickens.

Fungal antigens were also obtained from swabs taken from bathrooms or from the paint on walls.

Examole 4 Agcrlutination Test In order to determine the presence of one or more selected antigens in a sample, an agglutination test is carried out using the antisera. Before testing a standard or sample for immunoreactivity with antiserum A or B, the standard or sample is first subjected to a clean up process described below in order to prevent false positive results.

After seven days of growth, one loopful of each individual identifiable giant colony of fungus or sample was extracted in 1 ml of ethanol and then incubated at 370C for 10 min in a water bath with gentle shaking. (One loopful of fungus weighs between 2. 2 to 2. 8 mg (mean = 2. 5 mg)). The is ethanolic extract was then filtered using Whatman No. 2 filter paper into a glass tube. (11Whatmanll is a Registered Trade Marki. 50 gl (one drop) of this filtrate was placed on a glass slide and then the ethanol was evaporated by heating the slide at 370C. An antigen residue containing toxins was left as a spot on the slide. 50 M1 of sterile distilled water was added to the spot which dissolved the antigen residue completely.

Tylpe A Immunoprecipitation Test gl of Type A polyclonal antibodies was added to the 50 gl of test antigen on the glass slide. A white immunoprecipitate was formed immediately. On completion of the test a thin white film formed. When examined under the light microscope (40x) an immunoprecipitate was present which appeared brown and yellow in colour. Dilution of the antibodies A one in ten still gave an immunoprecipitate. No immunoprecipitate was formed when the antigen being tested was taken from a fungal colony of Fusarium or Penicillium. The results of the immunoprecipitation test with various standard fungal extracts is shown in Table 1 above.

A control test using a clean glass slide and 50 gl of water as the sample did not give any immunoprecipitate.

A 1:10 dilution of immunoprecipitate.

Type B Immunonrecipitation Test the antibodies gave an The procedure is the same as for the Type A immunoprecipitation test except that Type B polyclonal antibodies are used instead of Type A antibodies. The results of the immunoprecipitation test with various standard fungal extracts are shown in Table 2 above. With Type B antibodies any fungal spores present did not appear to interfere with the immunoprecipitation reaction. The size of the immunoprecipitate complexes appeared to be much smaller under the light microscope (40x) than the spore size of the fungi under test. When spores were present in the antigen sample the immunoprecipitate did not form around or on the spores. Antibodies B may have been directed against the mycotoxins of the fungi. The Type B immunoprecipitation reaction took place more slowly than the Type A immunoprecipitation reaction. Immunoprecipitation with antibodies B took a few seconds and required some gentle shaking in order for a white film to form. Fusarium antigens were found to react with Type B antibodies to give an immunoprecipitate. Under the light microscope the immunoprecipitate complexes with the various fungal antigens appeared bright yellow. The results of the immunoprecipitation test are set out in Table 2.

Penicillium antigens did not react with either of the antiserum to give an immunoprecipitate.

Exammle 5 The Detection of Fusarium in a Sample of Feed 0.1 gram of feed contaminated with Fusarium but not any of Aspercrillus, Mucor and Microsporum was extracted in 1 ml of ethanol by grinding in a pestle and mortar. Af ter shaking the sample vigorously the sample was incubated for 10 min at 370C with gentle shaking in a water bath. The incubated sample was filtered through Whatman No.2 filter paper into a glass tube. One drop of the filtrate was spotted on to a glass slide. The ethanol was evaporated off by placing the slide on a hot plate at 370C. After the ethanol evaporated a residue of antigens and mycotoxins was left on the slide. one drop (50 gl) of sterile distilled water was added to the spot and to dissolve the residue. The 50 gl sample on the slide was tested with antibodies A and B as described above in Example 4.

An immunoprecipitate formed with antibodies B but not with antibodies A. According to the response of the two sets of antibodies A and B, this test confirms that the feed is 10 contaminated with Fusarium alone.

Examiple 6 is The Detection of Fungal Contamination in a SamDle of Feed 1 gram of feed contaminated with various moulds was subjected to immunoprecipitation tests with antibodies A and antibodies B as described in Example 5. An agglutination reaction occurred with both antibodies A and B suggesting that the feed may contain one or more of Aspergillus, Mucor and Microsporum and possibly Fusarium and/or Penicillium.

Whilst the invention has been described with reference to the various examples, many variations and modifications thereof are possible within the scope of the invention.

Methods of raising antibodies described herein are claimed in Parent Application No. 9312370.1 from which this is a divisional application and methods of detecting the presence of particular antigens described herein are claimed in a further divisional Application No.

Claims (8)

1. Antibodies immunoreactive against one or more of Fusariu, Asipergillus, Mucor and Microsporum such that an immunoreaction takes place when the antibodies are challenged with one or more of their respective eliciting antigens.

2. Antibodies as claimed in Claim 1 not immunoreactive against Penicillium.

3. Antibodies as claimed in Claim 1 or Claim 2, wherein the immunoreaction is capable of being observed at least under the light microscope as an immunoprecipitate.

4. Antibodies as claimed in any one of Claims 1 to 3, wherein the antibodies are polyclonal antibodies and are specific to the said one or more of Fusarium, Aspergillus, Mucor and Microsporum.

5. Antibodies as claimed in Claim 3, wherein the antibodies are monoclonal.

6. Antisera containing antibodies as claimed in any one of Claims 1 to 4.

7. Antibodies as claimed in any one of Claims 1 to 5, conjugated to a protein, enzyme, radioisotope or chemical compound to enable the specific detection of the antibodies.

8. The use in an immunoassay or immunolocalisation technique for detecting the presence of selected fungal antigens of the antibodies claimed in any one of Claims 1 to 6.