The subject of the present invention is a diagnostic immunological preparation as well as its use in the detection and/or determination of the level of polyvalent antiphage antibodies. The preparation is broadly useful in the examination of the pathogenesis of infectious diseases and phage therapy. The increased interest in therapy using bacteriophages or proteins isolated from phages is due to their demonstrated efficacy in combating infections not treatable with antibiotics [1,2]. Bacteriophages possess a series of other advantages over conventional antibiotics, and primarily their specificity which makes it possible to destroy pathogenic bacteria without killing symbionts, thereby not affecting the subtle microbiological equilibrium of the organism [1,3-6]. The bacteriophage concentration in a patient's bodily fluids does not decrease, but is self-regulated. The number of phages grows exponentially in the presence of bacteria and rapidly drops in their absence. The selection of resistant bacteria is seen much less frequently, and moreover is resistance to a particular phage is observed, a different phage may be selected, which would be capable of infecting the resistant bacterium. They may also be used in parallel with antibiotics in combined therapy. Bacteriophages comprise a natural factor regulating the proliferation of bacteria in nature. For every bacterium there is likely a specific phage capable of infecting it, but of particular interest are the so-called polyvalent phages, specific to wide spectra of bacteria. The introduction of phage therapy into common use in medicine requires the design of methods of rapidly and effectively selecting appropriate phages, monitoring phage proliferation and penetration of a patient's organism as well as the release of toxins by the destroyed bacteria which may cause septic shock, and of phage removal from the organism, as well as the analysis of the presence of antiphage antibodies [7,8]. The occurrence of antiphage antibodies was noted during clinical trials prior to phage treatment [1,11-13]. These data, however, were not unequivocal due to the unavailability of a method of purifying and analysing the degree of purity of the phage preparations at the time [1,11-13]. The therapy was unsuccessful in some patients, which may have stemmed from the presence of lysogenic phages in the infecting bacteria, which were capable of inducing antibodies [H]. The presence of such antibodies may have a significant effect on the disease and the efficacy of phage therapy. Should the same antigenic determinants occur in the lytic phage used in the treatment and the lysogenic phage which immunised the patient, the expected therapeutic effect may not be reached. Because in the case of phages we are dealing with protein antigens, a single immunisation with a given phage may induce a long term response. It should be taken into account that lytic phages may be naturally responsible for cured bacterial infections. In such a case, the presence of serum antiphage antibodies may also be an effect of the disease, but there is no literature data on the subject. Patent application P 381 730 discloses procedures of the efficient proliferation of bacteriophages and their purification, which made it possible to obtain large quantities of pure phages with a high activity titre, without contamination with endotoxins and other bacterial components, which prevent a number of uses [9]. Further research has led to original observations which are the subject of the present application, relating to a diagnostic preparation and the determination of the level of anti-phage polyvalent antibodies using a specific immunochemical test.

The goal of the present invention is to deliver a specific diagnostic preparation for detecting and determining the level of polyvalent antiphage antibodies which, due to its formulation, lacks all of the drawbacks extant in prior art through this facilitates effective diagnostics and phage therapy.

The subject of the present invention is a diagnostic immunological preparation, characterised in that it contains a phage devoid of bacterial proteins, or immunoreactive phage proteins produced therefrom and devoid of bacterial proteins. Preferentially, the abovementioned immunoreactive proteins exhibit immunoreactivity during immunoblotting with human serum, plasma or other bodily fluids. More preferentially, the phage is a polyvalent phage.

The next subject of the present invention is the use of a phage devoid of bacterial proteins or immunoreactive phage proteins produced therefrom and devoid of bacterial proteins in the production of a diagnostic preparation for detecting and determining the level of polyvalent antiphage antibodies.

Preferentially, the diagnostic immunological preparation is used for immunochemical tests encompassing ELISA tests and is used to detect and/or determine the level of polyvalent antiphage antibodies in blood serum, plasma or other bodily fluids. Bacteriophages possess from several to a dozen or more types of protein which constitute its capsid. Phage proteins form morphological structures which make it possible to differentiate the head, sheath, base plate, tail fibres and etc. [3,4]. A number of these proteins exhibit enzymatic activity, i.e. that which is necessary to dissolve a bacterial wall or membrane. Thus, some purified phage proteins are also a potential bactericidal factor [5], particularly those from polyvalent phages with broad specificity. Polyvalent phages recognise receptors common to larger groups of bacterial strains such as teichoic acids, peptidoglycan, common core portion of endotoxins, colominic acid and other surface structures [14]. Research according to the present invention has shown that the level of antiphage antibodies in the serum is much lower than that of antibacterial ones, thus trace contamination with bacterial proteins is the cause of significant detection error. In light of this, purified phages are needed to determine antiphage antibodies or, most preferentially, proteins obtained therefrom or immunogenic protein fractions. To analyse their purity, we propose a comprehensive use of SDS-PAGE, immunoblotting and LAL/GLC-MS to analyse the purity of the protein fractions as antigens for the test. Research has also shown that individual human immunoreactive sera recognise several to a dozen or more proteins of a given phage, usually not main proteins, forming several reactivity patterns. Thus, the use of a single immunogenic phage protein as an antigen is incorrect, as it is necessary to use a set of phage proteins as antigens for the test. There are known solutions which detect and use one peptide or specific antibody, such as for Chlamydia, but these have a series of limitations aside from the aforementioned one. A solution like the latter is possible only for a highly specific system of a single pathogen, on the condition that one has access to a peptide specific for that phage and is exclusive of polyvalent phages. The result of further research using antiphage antibodies as a standard, produced using affinity chromatography on immobilised purified phage proteins, indicate the presence of cross- reactive antiphage antibodies necessitating the controlled selection of immunoreactive phage protein fractions as antigens and the essential standardisation of tests for detecting antiphage antibodies, which is the next novel element of our solution and confirms the need to use sets (fractions) of phage proteins in the preparation. Thus, the solution is based on the use of a pure phage immunogenic protein fraction as an antigen, the use of phage purity criteria proposed in the present solution as well as basing a test and its standardisation on reference antibodies produced for a particular test. During the initial stage, phage preparations were produced using known methods, such as the use of a detergent and ultrafiltration membranes [9] or, in the case when the detergent deactivates the bacteriophage, on an affinity column with immobilised polylysine [10]. The use of a large number of phage molecules makes it possible to separate their individual component proteins. Further, data obtained through an immunoblot with human sera made it possible to design ELISA tests which are used to quantitatively monitor the degree of immunization against bacteriophages. Due to the presence of antibodies against these components we observed in human serum, it is necessary to accurately and repeatably purify bacteriophage proteins in order to obtain trustworthy analysis results.

The present invention is based on the results of research on three polyvalent phages [14]: 1) specific for many serotypes of E. coli phage Kl, since colominic acid which constitutes the Kl antigen co-occurs with many E. coli O-antigens often noted in epidemiological reports, and is also a phage amenable to experimentation which facilitates the design of methods and tests, 2) polyvalent phage T4, capable of infecting a wide range of E. coli, Shigella and Salmonella strains, since its receptor is a conserved domain of an enterobacterial endotoxin, often classified in experimental therapy , as well as 3) polyvalent phage Hafiiia alvei, specific for the entire genus Hafnia. These bacteria are a frequent cause of many diseases. The purity of a diagnostic preparation according to the present invention is ascertained using chromatographic methods, using SDS-PAGE electrophoresis, endotoxin and bacterial cell wall component, as well as the LAL test and chemical analysis methods using the GLC-MS system. An immunoreactive phage protein used in a preparation according to the present invention is isolated, for example using preparative electrophoresis in the presence of dodecyl sulphate. A diagnostic preparation according to the present invention is used in an immunological test to detect and determine the level of serum antibodies against polyvalent phages, which play a significant role in the pathogenesis of infectious diseases. In order to prepare a diagnostic preparation according to the present invention, particular fractions of phage proteins are isolated, as well as using the whole purified phage, for a quantitative immunoenzymatic ELISA test to determine the level of anti-polyvalent phage antibodies. The protein fractions obtained from the phages were analysed using pooled human serum by immunoblotting so as to select immunoreactive fractions for the standardisation of the ELISA. Denaturing SDS-PAGE combined with immunoblotting was used to determine which of the phage proteins react with the antibodies present in the serum. Unexpectedly, full phage preparations were equally good antigens for the detection of anti-phage antibodies and were no worse than purified protein fractions. However, protein fractions comprise preparations with a defined molecular composition and an easily ascertained degree of purity. In turn, the whole phage comprises a complete antigen set and may be necessary for the initial determination and evaluation of the complete response of an organism to a given phage, which was often observed during the research. The purification of the proteins was also performed using routine chromatographic techniques such as molecular sieving, ion exchange and hydrophobic interaction chromatography. However, preparative electrophoresis turned out to be preferentially most comfortable. The present invention makes use of phage antigens and their protein derivatives to produce diagnostic preparations in a quantitative test for determining anti-phage antibodies in serum, plasma and other bodily fluids. Phage antigen analysis initially related to immunoblotting of human serum to determine the immunoreactivity of the produced protein fractions (Example 3), comparing them to fractions obtained via other means, meaning ultrafiltration in a saccharose gradient (Example 2). The purified proteins reacting with the antibodies used in the ELISA test are used in the quantitative analysis of human serum samples. A quantitative ELISA test was designed in which purified polyvalent phages or purified or cloned proteins, as antigens reacting with antibodies, are used to analyse the levels of antiphage antibodies. The use of phage proteins in the test made it possible to first discover antiphage antibodies in serum, since prior data [1,11-13] were unclear due to the lack of methods of purification and analysis of the degree of purity of phage preparations. Phage proteins produced via the present invention may be used in a series of other applications, in biotechnology and medicine. One should highlight as preferential the introduction of a test to detect antibodies against polyvalent phages, which is useful for monitoring antiphage antibodies before during and following phage therapy which is made possible by the proposed novel approach and the present solution. The possession of a method of quantitatively evaluating antiphage activity, proposed in the present invention, may also be used to enliven analysis standards for blood derivative preparations. The antibody analysis method will be used in monitoring and predicting therapeutic effects as an important factor in the prognosis. A high level of antiphage activity in healthy and ill persons makes it possible to evaluate which of the phages in question induce such a response, as well as being able to indicate a relation between an antiphage response and a disease, which may be useful in the future selection of phages for therapy. The production of novel, therapeutically useful phage strains, as well as the preparation of therapeutic preparations is many times cheaper than searching for novel antibiotics as well as the therapy using them. The solutions proposed in the present invention are simple, require no expensive reagents, and are based on three different preparations, making them applicable for a wider range of polyvalent phages. Very specific phages are of negligible utility, due to the large variability of the strains, whereas polyvalent phages are very valuable due to their presence in nature, in living organisms. This is also the reason for the presence of antibodies against polyvalent phages in the human population observed in our research. Due to the clinical importance of these antibodies, one should stress the practical value of a diagnostic preparation being the subject of the present invention . The present invention has been demonstrated in examples not exhausting the scope of its protection.

Example 1. Preparative electrophoresis of T4 phage proteins

The isolation of T4 phage proteins was performed using preparative electrophoresis in the presence of SDS (2 cm 5% stacking gel, 4 cm 8% separating gel and 6 cm 10% separating gel) under denaturing conditions. Electrophoresis was performed using a Pep Cell model 491 apparatus (Bio Rad), in a tube of 37 mm diameter and 12 cm gel length. The stacking and separating gels were poured and the tube was left 4°C overnight. The gel was loaded with the T4 phage protein preparation purified using the PDL method [9], which contained 25 mg total protein. The lyophilised preparation to be separated was dissolved in ImI of protein denaturing buffer and boiled for 15 minutes. After loading, electrophoresis was performed over a 24h period at an appropriate voltage. 1.5 ml fractions were collected at a rate of ImI/ 1.5 minutes. After 1O h the amperage was changed from 40 mA/257V to 74mA/300V. The protein was eluted with water and 280 fractions were collected. Every fourth fraction was collected for analysis, in order to pool those which showed the same electrophoretic banding pattern. Overall, 15 pooled fractions were collected, which were analysed using denaturing electrophoresis. T4 phage protein preparations collected following preparative electrophoresis were dialysed against water, lyophilised and dissolved in ImI mili Q water, and then their concentration was determined. The protein fractions were analysed using immunoblotting with human sera in order to determine the presence of immunoreactive protein bands and to determine the utility of a given protein fraction as a diagnostic factor in an immunochemical ELISA test, in order to quantitatively determine antiphage antibodies in bodily fluids and biological preparations.

Example 2. Isolation of T4 phage proteins through ultracentrifugation in a saccharose gradient

In order to separate individual T4 phage proteins we also used ultracentrifugation in a saccharose gradient as a reference method on the laboratory scale. A saltatory saccharose gradient was prepared in tubes : 40, 31,75, 22,5, 13,4 and 4% (w/v). The gradient was settled overnight at 4°C. The amount of purified phage was 1.0 x 10 ¹⁰ pfu/ml in 1.5 ml, or 1,5 x 10 ¹⁰ phage molecules, which constituted 1.08 mg total protein. Two tubes with saccharose were loaded with 0.75 ml of phage preparation and ultracentrifuged in a Beckmann L7-55 ultracentrifuge with a 70Ti rotor, for 5 hours, at 50 000 RPM, at 5°C. 45 ImI fractions were collected and dialysed against water. The fractions were analysed using SDS-PAGE electrophoresis and fractions with identical banding patterns were pooled. In all, 7 pooled fractions were collected. Analysis of the ultracentrifuged T4 phage preparations produced via ultracentrifugation in a saccharose gradient made it possible to partially separate individual phage proteins. Protein concentration in the preparations was determined using the Lowry method. Moreover, SDS-PAGE electrophoresis in denaturing conditions in a 12.5% gel with 20 μg of loaded protein confirmed the purity of the phage and an absence of contamination with bacterial proteins or endotoxins. Example 3. Immunoblotting of phage protein fractions with human serum. Immunoblotting was performed following SDS-PAGE using gels maintained in transfer buffer for 15 minutes. Immobilon-P membranes were soaked for 15 s in 100% MetOH, 2 minutes in milli Q water and 2 minutes in transfer buffer. The transfer of separated proteins was performed for 1 h at 100 V in a transfer cell [BioRad]. The membrane was stained afterwards with Ponceau S, and then destained in milli Q water until pink colouration disappeared. The membrane was dried on Whatman 1 tissue and stored. The dry membrane was soaked in 100% MetOH and rinsed in milli Q water and then blocked in 1% BSA in TBS-T (5 ml) (TBS + 0.05% Tween 20 with 0.5 ml 1% BSA) at 37 ⁰ C for 1 h, whereafter the membrane (Immobilon-P) was rinsed in TBS-T 1 x 15 minutes and 2 x 5 minutes. Next, the Immobilon-P was incubated with a solution of pooled human serum diluted 25Ox (5ml TBS-T z 1% BSA + 20 μl serum). The initial phase of the incubation took place in 37 ⁰ C for 1 h with shaking and then 4 ⁰ C overnight. Unbound antibodies were rinsed off with TBS-T (1 x 15 minutes and 2 x 5 minutes). A solution was prepared of goat anti-human IgG conjugated with alkaline phosphatase (0,5 μl antibodies in 10 ml TBS-T with 1% BSA) and the membrane was incubated therein at 37 ⁰ C for Ih. Excess antibody was rinsed off with TBS. The image was developed in a solution of 5 ml TBS-Mg ²⁺ buffer with 50 μl BCIP (30 mg/ml in 70% DMF) and 50 μl NBT (15 mg/ml in 100% DMF). Example 4. Determination of antiphage antibody levels in human serum.

The level of antiphage antibodies in human blood serum or plasma was measured using an ELISA test, in plates coated with a purified polyvalent phage preparation or a protein fraction containing immunoreactive proteins. In order to avoid cross -reactivity each sample was tested in a reaction with serum protein or whole serum, whereafter control values were separated. The plates were coated with a T4 phage preparation or a derivative fraction containing immunoreactive proteins (1 μg/well, 2.7 x 10 ⁴ pfu/well, in carbonate buffer pH 9.6). The plate was blocked and rinsed thrice with TBS-T (245 μl /well). Human serum diluted 25Ox was loaded at 100 μl per well in 4 replicants and incubated for an hour at room temperature. Next, unbound antibodies were rinsed off (3 x 245 μl TBS-T/well) and each well was loaded with 100 μl goat anti-human IgG conjugated with alkaline phosphatase (1:30 000). The plate was incubated for an hour at room temperature and then rinsed thrice with TBS-T (245 μl/well). Substrate was added for the alkaline phosphatase (p-nitrophenyl-disodiumphosphate) at 1 mg/ml in carbonate buffer with magnesium and chloride ions, at 100 μl per well, or lOOμg substrate. The reaction was stopped by adding mili Q water. Reaction intensity was measured by measuring absorption at 405 nm using an Opsys MR plate reader (Dynex Technologies). The level of anti-phage antibody levels in the patients' sera was expressed as the average absorption on the plate at 405 nm after subtracting the average absorption at 405 nm of the control samples. Bibliography

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