NL8520062A - BACTERIOPHAGES AS RECOGNITION AND IDENTIFIER. - Google Patents

Description

β 5 2 O O 6 2! - Bacteriophageii as recognition and identification means - j i; i ·.

i; | The invention relates to the field of immunology, provides a new and reliable method : for the identification and determination of materials of both; procariotic and eucariotic cells, and includes a selected bacteriophage linked to a visualizing agent.

Furthermore, a research package is provided for the smooth determination of bacteria, eucariotic cells and various molecular materials.

Currently, almost exclusively antibodies are used to identify molecular and cellular structures that do not otherwise reveal themselves.

! For example, they are used to distinguish one protein from another, one polysaccharide from another, or one cell from another. The interaction between the antibodies and the "identified" structure can be determined by a number of different methods aimed at revealing the presence of the antibodies.

In some experiments, an antibody bound to the structure, cell, or solid phase to be identified, becomes 20.

| made visible immediately. More often, a second antibody becomes:

i visualized and directed against the first antibody. Top I

In addition, protein A of Staphylococcus aureus, Cowan I strain, S

| visualized, after which it binds to the first antibody. Another group of reagents used to test 25. . ! making antibody visible is represented by biotm; and avidin which recognize each other and can be used together with antibodies. Fluorescent material, enzymes, radioactive

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I materials, large particles, such as erythrocytes, or granules are i

used to make the antibody visible directly or indirectly

! 30! to make. ; 8520062

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Each of these methods has shortcomings j and limitations which can be explained using! the use of fluorescent materials for visible j

making the antibody. If the amount consumed by a cell; 5 bound antibody is too small, the fluorescent I.

I do not detect reagents. This causes analytical problems, particularly when using dual laser flow cytometry or fluorescence assay systems. Furthermore, the fluorescence obtained with an antibody is! 10 system on tissue parts not very clear. This problem can be overcome by using enzymes that are coupled to the antibody. However, a number of steps are always required! i in the staining process.

| Solutions to the problem have come down to using fluorescent bacteria or fluorescent bacteria grains. The former can only be used to a limited extent in fluorescence analysis due to problems in removing! unbound excess bacteria and also due to a significant change in the light scattering profile of the cell surface, whereby flow cytometry cannot be used appropriately. The fluorescent beads tend to stick to cells, are difficult to produce or expensive, and they are also large enough to cause changes in the light scattering profile of the cell surface. Moreover, in fluorescence assay systems requiring filtration j and washings, the excess particles or bacteria j are difficult, if not impossible, to remove. The use of reagents such as bacteria or colloidal gold for colored,; ! . . : fixed cell preparations are limited to microscopy. Consequently, a need has arisen for another group of reagents, which! | combines the advantages of antibodies with those of particles and j which can be used for flow cytometry for tissue parts i or for solid phase assays for soluble materials.

i | l i 8520062 'V' ί - 3 - j

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j Solid phases used in packages for determining antigens or antibodies in solutions can be used for this either directly or in competitive assays.

It has not been found possible to have a reagent that has all 5 advantages of antibodies, such as penetrability and specificity; combined with that of particles, such as visibility.

In fact, there was a need for a reagent that acts as an antibody, but is much larger, though not so large, as to hide the structures to be identified. Such a reagent must remain from some structures, such as fixed cells or tissues, and excess reagent must be easily removed.

It is likely that bacteriophages have been found to be either too small to serve as effective carriers or to trap in a non-specific manner, particularly due to their cilia. The only thing bacteriophages have been used for in identification experiments is that they have been used as viruses for their bacterial host i 'i or antigens have been applied to their surface, i usually by a mild chemical coupling method, and j; 20 subsequently used antibodies against these antigens to infect the ability of the bacteriophage | bactetia in | neutralization systems for bacteriophages. For example, bacteriophages were used as living viruses only in Haimovich U.S. Patent 3,171,705, et al., I 25, and Young U.S. 4,104,126. The; bacteriophage was not used as an antibody support ! as an identification and staining reagent. It is likely, j | that an expert has been detained for the reasons given above j; | | of using the phage for any other determinations with j | 30 antibodies. In U.S. Pat. No. 4,282,315, j Luderer, et al, suggested using radioactively labeled animal viruses j to detect receptors on materials that | natural receptors of these viruses are or mimic such receptors. In essence, the animal viruses were selected

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because of their natural affinity for certain receptor materials, j after which they were removed and multiplied. This was essentially designed either to identify virus receptors or for hemagglutination purposes, that is, there was a clear correspondence between the virus receptors and the identified receptors. Bacteriophages, which are bacterial viruses, have not been included in this category for these obvious reasons. In the first place, they are not used for any determinations of the hemaggluti-; nation or of the inhibition of heme agglutination; these provisions; are based on characteristics of only certain animal viruses.

| Secondly, bacteriophages are known to be specialized. have structures, usually in the form of a tail, that are used to recognize the receptor on bacteria, leading to infection. It is known that once this tail binds to its receptor, even when it is not on bacteria but in solution, the nucleic acid is expelled and the virus becomes non-infectious. Such a selection method could not possibly have been eligible according to the methods of the Luderer, et al. Patent. On the other hand, the bacteriophage would not have been eligible for selection: | regarding binding to the protein of the head, since i '; | mutations in the proteins of the head are considered lethal to the bacteriophage. This may explain why the Luderer, et al. Patent does not consider the bacteriophage as a possible alternative. , despite the fact that bacteriophages have many benefits can be produced in copper and are easily formed in a very large quantity, for example orders larger than animal ones! JU j | viruses.

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; The invention relates to a new method for the identification and quantification of molecular; and cellular materials of both procariotic and eucariotic 8520062! - 5 -

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I cells, wherein a test sample is combined with a selected bacteriophage under binding conditions to obtain a conjugate phase in the test sample, which conjugate comprises the bacteriophage coupled to the molecule or cells attempted to be identified. A tb annealing agent is incorporated into the bacteriophage before or after the binding step to greatly improve recognition of the test material in conventional analytical assay methods. In carrying out the invention, the bacteriophage can be selected to bind with the head or tail. In the | last case, this is brought about by "external imaging" where the bacteriophage is modified such | that it behaves like an antibody.

In general, mutants of the bacterio-I15,. . , | phage used. The method of the invention can be made suitable for the analysis of proteins, carbohydrates,; lectins, bacteria of different types, pathogens and mixed molecular or cellular materials present in tissues, cells or fluids.

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Furthermore, it is an object of the invention! provide test kits comprising the selected bacteriophage and suitable test agents to provide the test material in a form suitable for use in any chosen analytical tool.

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The recognition of a structure by a

means of identification such as an antibody can be subdivided I

den in two parts, the "recognition site" (intelligence), that; | ie the specific place that binds a complementary structure j, and the "visualizer1! 1 (visualizer), that is! i 30 | the structure that is somehow visualized. j

For example, in the case of fluorescent bacteria, \ ί! beads coated with antibody, the "recognition site" i provided by the attachment site of the antibody molecule and the "visualizer". through the rest of the molecule with the 8520062 t: I - 6 - i molecular attachments (fluorescent, radioactive, etc.).

To provide an additional "visualizer" according to the In the invention, the antibodies are coupled to bacteriophages.

This coupling can be covalent, such as with glutaraldehyde or bifunctional reagents, or non-covalent, such as with hybrid antibodies. Also, according to the invention, the bacteriophages can be selected and constructed to provide the "recognition site", that is, to function as antibodies, and also carry the "visualizer". : 10 ...

In the identification of bacteria m patholo- | gic liquids takes time to culture the bacteria and | to identify and, in many cases, the bacteria not be cultivated in culture. For these cases be! bacteriophages visualized and either coated with antibodies 115 | or bound naturally through their receptor.

| In carrying out the method of the invention, bacteriophages are used as the visualizer, or carrier of the recognition site, which is provided by the antibody. Antibodies can chemically attack bacteriophages 20 ....

coupled with bifunctional reagents such as glutar; aldehyde or other covalent or non-covalent coupling agents; being used. Bacteriophages are taken with avidin or biotin; | coated to the antibody containing biotin or | avidin to bind. The bond is obtained via! 25 ...

| biotme avidin recognition. j] Bacteriophages serve as visualizers by j i .... i | to bind the structure to be identified using hybrid:! antibodies. These are placed with an attachment site against the bacteriophage and with the other against a first antibody or. . . . . i target the structure to be identified. The bacteriophage can also be linked to lectins or carbohydrates for recognition of the respective complementary structures, i.

The visible maker provided by the bacteriophage is made using fluorescent dyes, other i.

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dyes, radioactive isotope material, enzymes, or metals such as silver or gold. The bacteriophage can also be manipulated to contain the appropriate enzyme. | - * Bacteriophages can be selected such; that they provide both the recognition site and the visualizer by genetic engineering, which provides them with an "attachment site": After mutation, the bacteriophages are then selected for the property of the head on molecules j 10 such as immunoglobulins or to bind to glycoproteins or proteins of animal cells The mutants are obtained, for example, by ultraviolet irradiation of both bacteriophage and bacteria, followed by culture of the bacteriophage.

The bacteriophage is collected, purified and the selection pressure is applied, viz., Binding to cell surfaces or to solid phase-coupled molecules. The bacteriophage becomes immediately or after the cell has been treated with the antibody | recognized by the bacteriophage rendered fluorescent 1 for cell surface identification. The lethal nature of head mutations is avoided by using very large numbers of particles and selecting temperature resistant mutants; whereby selection is therefore made on very rare cases! | which are still compatible with the survival of the bacteriophage.

Another set of mutants can be prepared j

; 25 by making use of the possibility of the tail to I.

to bind to a specific structure on the surface of bacteria. Bacteriophages are selected by mutation and selection, which can recognize certain structures with their tail. | I This is done using "external imaging"; ! 20 (external imaging), an unobvious analogy with; ; internal imaging m 1 the anti-idiotype network of antibodies, as discussed by Urbain, et al., j | 8520062-8 -:; j f: j Progress in Immunology, 1980, Academic Press, Vol. I p. 81-92.

| The molecule of MX "to be identified is injected into an animal and antibodies are made against it. The purified anti-" X "antibodies are coupled to a solid phase and treated with a very large number of bacteria. These bacteria are extracted from a bacteriophage sensitive parent strain selected for resistance to bacteriophage "Y" Those against bacteriophages: resistant bacteria have no receptors for the bacteriophage Bacteria that bind to these antibodies are selected and grown to have a structure that mimics "X". The selection of bacteria that can exhibit "X" -like structures | can be tested by their ability to bind purified anti-"X". It is very unlikely that such a! structure may be the receptor for the bacteriophage.

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These bacteria are then mixed approximately equal parts with the bacteriophage sensitive bacteria which had been irradiated with UV and treated with mutagenized (for example UV irradiated) bacteriophage. The mutant bacteriophages that emerge from susceptible bacteria and which infect the receptors. .

can recognize bacteria that are resistant to bacteriophage, grow in these bacteria. Therefore, all the lysis will be cloudy since only the bacteriophage sensitive bacteria are lysed, except for a few clear spots, in which both bacteria are lysed as a result of the mutant. The spots are removed with a; absorbent material and the mutants recognizing "X" are detected with a "^" sample made visible, i.e., radioactive, fluorescent, etc. The mutant bacteriophages are detected on the plate and on their new bacterial host cloned The ability of all clones to recognize "X" is determined on a solid phase by competitive tests with free "X" and competition by "X" for the infection of bacteria by this mutant bacteriophage Although the recognition of the receptor on bacteria by the tail of the receptor may have some similarities with antigen-antibody interactions, the recognition process with the bacteriophage has its own other characteristics, which makes them surprising. and makes unexpected replacement for antibodies 5. It is also unexpected that the bacteriophages are selected for the desired site recognition, as it is known that the mutations of the bacteriophage are host-related gentleman's range are fairly frequent. j Using other genetic manipulations, the bacteriophages are induced to recognize a certain antigen. Bacteria are used that have certain surface structures that are controlled by plasmids.

For example, the gene for nX "is introduced into the plasmid. Bacteria displaying MXM on their surface are then selected as described above. These surface proteins or glycoproteins are located on structures used by the bacteriophage as receptors. By selecting bacteriophages recognizing these structures as their infection receptors, bacteriophages recognizing MXM are obtained.

Consequently, an external image of the antigen on bacteria 1 is obtained. j

In another approach, the tail of the bacteriophage is made with the same sequences as heavy! ! and / or light immunoglobulin chains, by recombination with immunoglobulin genes in plasmids. Original bacteriophages with the normal tail sequences are removed by absorption with natural hosts. These are mutated and selected for recognition of MX by the methods described above. A complete heavy and light chain device is obtained by simultaneously infecting bacteria with "X" on the surface with two bacteriophages. one of which presses V gene gene and the other expresses V gene products. [8520062 ......!! ... ......... .. i

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| An agent for carrying out the method of the invention is tantamount to assembling a package comprising an appropriate bacteriophage which is coupled with a visualizing agent for use in the identification of bacteria, eucariotic cells and other molecular materials. A number of portions of the phage can be provided, each in an amount such that it is effective in the intended experiment.

The following examples serve as an explanation,! 10 without limiting the method of the invention to this.

Example I ~ A

Bacteriophages are coupled to monoclonal or I polyclonal antibodies after they are fluorescent or 15 have been made radioactive, or coupled to peroxidase, and are used as a colorant, or identifier. To determine murine immunoglobulin G (MIG), the bacteriophage is coated with anti-MIG antibodies by using a coupling agent, for example glutaraldehyde or another bifunctional reagent. The phage becomes fluorescent

made by coupling with fluorescein isothiocyanate, rhodamine j or other fluorescent dyes, by treatment with ethidium bromide, which binds to the DNA of the phage, or by other dyes that bind to nucleic acids. The phage is made radioactive or by incorporating radioactive I.

Materials in the protein or nucleic acid or by conventional methods of coupling radioactive materials to proteins. In the alternative, a metal such as silver is added to the phage by conventional methods. j; The bacteriophage suspension was prepared as follows:;

In a test example, bacteriophage T4 was grown in YS57 strain of Escherichia coli (Trp, Pro, His) ♦ Bacteria were grown 8520062! - 11 - I grown in tryptic soy broth (DIFCO) and phage mixed for a wide range of infections from 0.5 to 1. It; mixture of bacteria and bacteriophage was incubated in soft agar on a nutrient layer of hard agar. After overnight incubation, the bacteria were completely lysed by the phage and the top layer of soft agar was collected and with chloroform! and treated with 0.01 M EDTA to precipitate the non-phage material.

After 1 hour at 37 ° C, the mixture was stirred at 5000 G for 10 µl minutes and the supernatant was collected. '10. , j! To remove free nucleic acids, the above j

| liquid at 37 ° C for 1 hour treated with 20 µg / ml I.

DNAase and 20 µg / ml RNAase. To wash the phage, NaCl and j I j j polyethylene glycol (PEG) were added to final concentrations j of 0.5 M and 6%, respectively, and the mixture was washed for | , 15 ^ | Incubated at 4 ° C for 18 hours. · After centrifugation at 8000 G for 30 minutes and resuspending in 0.14 M NaCl 2, an ex-bacteriophage concentration of about 10 infectious units / ml was obtained. The absorption profile at different | wavelengths were that of a pure phage population.

I 20! Before coating with antibodies, the vol. '

| method. The phage was slurried in 0.1 M

1 i i,, | phosphate buffer at pH 8.5 mixed with glutaraldehyde to a concentration of 1% and incubated at 25 ° C for 1 hour.

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I Excess glutaraldehyde was removed through the 0.5 M phage

Precipitate NaCl and 6% PEG and resuspend in buffer at pH 8.5. Antibody, anti-rabbit Ig was added to give 1 mg of antibody / 1 mg of phage protein.

After 2 hours incubation at 4 ° C with rabbit lymphocietan, the cells were 30 ...

washed three times by centrifugation at 900 G for 5 minutes.

The binding of the fluorescent phage was compared to that of the fluorescent phage coated with either normal IgG instead of antibodies or fluorescent 8520062; - 12 - ran anti-Ig antibodies. The cells were evaluated under the fluorescence microscope. The macrophage character cells (i.e., large) contain 2-5% phage particles in both normal Ig and anti-Ig antibody preparations. However, of the cell preparation I5 treated with phage coated with anti-Ig antibody, 47% of the cells had surface fluorescence, | while the cells treated with normal Ig-coated phage I had only 1-2%.

The surface fluorescence was more intense than that of the same cells which fluoresced with anti-Ig

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! antibodies were treated. Therefore, the anti-Ig antibody-coated phages showed binding specificity and gave a j

intense fluorescence. I

| Example I-B j | .............

| Concentrated, purified bacteriophage is treated with a heterobifunctional reagent. Anti-allotype- j | rabbit antibody (anti b ^) is thiolated and bridged between the two is formed so that the rabbit's antibody | 20 is coupled to the phage. As a control, the phage is then made fluorescent. The phage-antibody complex is then continued. mixed with the cells, washed and examined with a microscope or other suitable instrument. The degree of fluorescence is determined by the amount of use of amino groups or phage 25 proteins.

Example I-C

The antibody-coated phage is used to treat fixed tissue parts. A lymph node section is treated with a fluorescent phage preparation, washed and examined under UV light. In an alternative method, coated phage are disclosed by addition of the substrate at the end 8520062-13 by standard methods. This method is also used for phages selected to bind spontaneously.

5 Example I-D

The phage selected to recognize MIG either tail or head is mixed with a solution containing MIG antibodies, e.g. monoclonal antibodies. This phage is fluorescent or radioactive.

After washing by precipitation with PEG, it is used to replace phages chemically coupled to antibody. The phages are visualized by an appropriate coupling method.

Example II

Bacteriophages are intended for use in the rapid diagnosis of pathological materials containing bacteria. Synovial fluid or exudate is streaked, fixed, phages are added, and their presence is visualized with suitable instruments, such as UV light for fluorescence, bright field for enzymes, etc. Using automated computer-connected scanners makes the method very made quickly. Binding of phages by anti-phage antibodies is avoided by competitive saturation with free phage protein or by treating the sample with formaldehyde. Very small numbers of bacteria, even when dead, are easily identified. Rare microorganisms can be identified by adding two-step complex phage suspensions against a wide variety of pathogens, followed by separate suspensions in those products that prove positive.

To mimic the conditions in the identification of bacteria in pathological samples, the following 8520062-14 experiment was performed.

T4 bacteriophages were grown and purified as in Example I-A. Different amounts of fluorescein i

isothiocyanate (FITC), from 0.5 mg to 8 mg, were added per 1 mg of 5 phage protein. The phage suspension was diluted with 0.1 M

adjusted to a pH of 9.3. FITC was added with stirring at 4 ° C, then stirring continued for 2 hours and! then incubated at 4 ° C for another 20 hours.

Excess FITC was removed by dialyzing the suspension against 0.1 M phosphate buffer (pH 8.5) for 3 days at 4 ° C. The final molar ratios of FITC / protein in seven preparations were (1) 3.8, (2) 8.0, (3) 11.3, (4) 18.6, (5) 29.2, (6) 19, 3, and (7) 16.4.

To determine the binding specificity and the ability to reveal the presence of a microorganism in a field, the T4 phage grown in E. coli strain YS57 were tested for binding to YS57. As controls, E.coli USC 106, which is an anti-phage resistant mutant of YS57, Bacillus globiggi and Salmonella Schottmulleri were used. To test the binding, phages were mixed for 10 minutes at 25 ° C with bacteria fixed with formaldehyde, then washed three times to remove the unbound phage.

The phage sensitive E. coli YS57 became intensely fluorescent so that even a microorganism could be clearly seen under the fluorescence microscope. Phage preparations (1), (2) and (3), i.e. up to a molar ratio of FITC / protein of 11.3, were made intensely fluorescent, and the mutant USC 106, B. Globiggi and S_. Schottmulleri not.

Therefore, the phage specificity was maintained at a molar ratio of 11.3, which is equal to the molar ratio commonly used for antibodies. However, since the phage particles have about 500 times more protein than antibodies, the phage particles also provide that much greater total fluorescence.

When fresh USC 106 and YS57 strains of E. coli were used (i.e., not fixed with formaldehyde), the phage bound equally to both bacteria.

5 This was an unexpected finding, showing that the preparation must first be fixed with formaldehyde. This gives it! advantage of destruction of antibodies, in particular natural antibodies, which may be present in pathological preparations to be investigated and which can bind the phage.

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Example III-A

The first condition for obtaining mutants of phages that recognize molecules and cells is that the phage does not already naturally bind to such cells or molecules. Thus, phages as in Example I-A were prepared and fluoresced to a molar ratio of FITC / phage protein of 11.3. These phages were tested for their ability to bind to complex cells, human lymphocytes treated with monoclonal mouse antibodies, and rabbit lymphocytes. Human raononuclear cells were purified from peripheral blood using the Ficoll-hypaque method, washed, and incubated with mouse monoclonal anti-human T cells at 4 ° C for 1 hour, and washed again.

Rabbit lymphoid cells were obtained from spleen and lymph nodes using conventional methods, mixed and washed.

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Suspensions containing 10 cells / ml (human or from the 12 mouse) were treated with 10 fluorescent phage particles, which were prepared as in Example I-A but not antibody-coated. After incubation for 1 hour at 4 ° C, the cells were washed and examined under the microscope.

No surface fluorescence was observed, indicating that T4 bacteriophages, which are made fluorescent 8520062 - 16 - do not adhere non-specifically to MIG, human | lymphocytes or rabbit lymphocytes. This surprising observation provided the basis for selecting for bacteriophages that bind.

5 The selection of phages that other! bind structures than they naturally recognize, is used up! carried out in two ways: by subjecting different existing bacteriophages from different pools to a comparative study and by synthetic manipulations. An example of a comparative study is the following.

Bacteriophage T4 does not bind to fresh suspensions of human or rabbit lymphocytes. To determine whether other structures of the blood components can be identified, swabs were prepared from rabbit and human blood cells and subsequently treated with formaldehyde. The smears were then treated with a 13 suspension containing 10 T4 phage particles per ml for 30 minutes. made fluorescent by treatment with FITC. The smears were washed, a cover was applied and examined under a fluorescence microscope.

Very intense fluorescence was only seen in the cytoplasm of the leucocytes. The core, membrane and red blood cells were not fluorescent. This test shows that somehow, through an unknown and not obvious mechanism, a structure in the cytoplasm of the white blood cells is recognized by the FITC-labeled T4 phage.

The selection of bacteriophages is also carried out by synthetic manipulations. In the preparation of bacteriophage mutants for hst recognizing mouse immunoglobulin (IgG), Escherichia coli bacteria, strain YS57, are prepared in petri dishes, as in Example 1A, and irradiated with UV light 8520062-17 - so that 50-90% of the bacteria is killed; this step is performed to initiate the DNA repair mechanisms. A suspension of T4 bacteriophage is also treated with UV light to kill about 80% of the bacteriophages and cause mutations. Bacteria are infected with the bacteriophage and the bacteriophage is grown, collected and treated with chloroform. The phage suspension then becomes; concentrated by precipitation with polyethylene glycol or by ultracentrifugation to obtain a suspension with more than 10 10 infectious units / ml.

The bottom of a plastic petri dish; is coated with mouse IgG (MIG) directly by incubation at 25 ° C and pH 9.2 overnight, or by using poly-L-lysine as a coupling agent. This MIG has no anti-phage antibody activity, ie it is either monoclonal for a different specificity or it is pre-absorbed with phage or only the Fc portion is used. The phage is added to the dish and incubated at 37 ° G for 1 hour. The plate is carefully washed to remove unbound phage. To improve the chance of obtaining the mutants, mouse IgG is attached to particles, erythrocites, bacteria or granules, either chemically or by the antibody function.

The plate which has been phage treated and washed is used to grow the phage directly in bacteria by adding bacteria in soft agar. The plate is first repeatedly washed to remove the non-specifically bound phage. To increase the chance of obtaining head mutations that survive, the phage are also grown at higher temperatures, for example 42 ° C. The phage colonies are removed and grown in susceptible bacteria and cloned again 2-3 times.

As a preliminary study for phage clones that recognize MIG, the following method is used. Plates are coated with MIG as described above, one parent strain of the phage is added to several plates in addition to the mutants, incubated and washed, and finally bacteria are added. Although the parent strain gives very few spots, the mutants that bind to MIG give many spots and even confluent lysis of bacteria.

Example III-B

In a method related to that of Example III-A, the phage colonies are recorded on an adsorption paper. Radioactive or fluorescent MIG is added, incubated to promote binding of MIG to the mutant, then washed and then examined with a radioactivity scanner and UV light, respectively. The affected mutants are returned to the original gel and cloned. For additional evidence, all colonies formed are collected and grown in bacteria, each clone in two tubes, one of which contains u-labeled amino acids. The phages are collected, treated with chloroform, collected by precipitation with polyethylene glycol or by ultracentrifugation, and added to MIG-coated microwells.

After incubation at 37 ° C for one hour, the wells are washed, sodium decyl sulfate (SDS) solution is added to remove bound radioactive material and the ppm.

determined. The phage clone giving a larger count than the background is then grown and tested again for the ability to bind MIG in the same system as above.

Example III-C

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The bacteriophage that shows binding to MIG is visualized using one of the usual methods. The bacteriophage is rendered fluorescent by coupling with the fluorescein isothiocyanate, rhodamine or other fluorescent dyes, by treatment with ethidium bromide, which binds to the phage DNA, or by other dyes that bind to nucleic acids. The phage is made radioactive either by incorporation of radioactive materials into the protein or nucleic acid or by conventional methods of coupling radioactive materials to proteins. In an alternative, a metal such as silver is added to the phage by conventional methods.

To test the value as a reagent of this phage, human lymphoeites are treated with MIG anti-human T-cell monoclonal antibodies. The cells are washed and then, for example, the fluorescent phage is added. As a control, the fluorescent phage is added to human lymphoeites that have not been treated with the monoclonal antibody. After the 15 lymphoeites have been treated for 30 minutes, they are washed and the cells are examined under the microscope with UV light. The intense fluorescent staining of 80-90% of the human peripheral blood lymphocytes shows that the phage recognize mouse IgG on the surface of the lymphocyte. Failure to fluoresce cells treated with parental phage instead of the mutant or previously treated with normal MIG instead of anti-T cell antibody gives a control. Another control is given by the fact that purified human B cells do not become fluorescent when treated as the: 2B unseparated peripheral blood lymphocytes.

Example IV

The selection of mutants by external imaging requires the preparation of bacteria that have phage receptors that mimic a particular identifiable macromolecule, eg mouse IgG (MIG). In this case, MIG is injected into rabbits or rats 8520062-20 to induce anti-MIG antibodies.

The following method aims to make a bacteriophage act exactly like an antibody by using its tail as an attachment site.

1) The anti-MIG antibody is combined with affini-;

purity chromatography and coupled to a solid phase, such as the bottom of a petri dish, by poly-L-lysine. _E. coli. resistant to the phage are grown, washed, suspended in physiological buffered saline, added to the Petri dish and at 4 ° C for one hour

incubated. The Petri dish is carefully washed and culture medium is added. Bacteria that grow are exposed to the same binding cycle 4-5 times, each time in the presence of the phage to maintain the phage-resistant character of the bacteria. At each exposure, the bacteria are treated with mutagens to increase the number of mutants.

2) Bacteria selected are cloned and each clone tested for binding by the anti-MIG antibody by conventional methods. The bacterial mutants grow to a greater degree than the parent strain, if they are allowed to attach to the bottom of the plates coated with the anti-MIG antibody, thereby giving the mutant a selective advantage.

3) The phage sensitive bacteria are UV irradiated, the phage are UV irradiated and then mixed with phage sensitive bacteria for infection and mutant formation. However, the phages and bacteria can also be mixed first and then irradiated with UV. These bacteria are mixed in equal proportions with phage-resistant bacteria which are recognized by anti-MIG antibodies. The phage lyses all phage-sensitive bacteria, but does not attack phage-resistant bacteria. When a mutant phage, which recognizes the MIG-like structure on the phage-resistant bacteria 8520062 * - 21 -, a bright spot is formed.

4) The clear spots are collected, grown in bacteria obtained in step 2, and each clone is tested for its interaction with MIG with conventional competition or binding assays. The clones are tested for their ability to infect their host in the presence of MIG which has no antibody activity against the phage. When the phage recognizes MIG with its tail, the phage binds to the - MIG and can attach itself to its host. , As a result, no lysis takes place. The precise recognition site is determined by using fragments of MIG.

The binding is tested on a solid phase coated with MIG, followed by anti-phage antibody labeled with peroxidase and substrate. The characteristic color change only appears where the phage is bound and shows its presence. The phage are visualized as in Example I.

Example V

Some surface receptors, or antigens, on bacteria are controlled by plasmids. An example is the sexpilus on E.coli. This pilus is recognized by sex-specific phages, such as 0X174. The MIG heavy or light chain constant region gene is included in the plasmid controlling the sexpilus. Bacteria that express MIG instead of or in conjunction with pilus proteins are selected using a solid phase with anti-MIG antibodies. Phages that recognize this pilus are then selected and shown to recognize MIG as before. In the first step, the phage-resistant bacteria are selected by the positive pressure of the anti-MIG antibody. The phage are visualized as in Example I.

8520062 - 22 -

Example VI-A

Bacteriophages are grown in bacteria that have variable genes for an anti-MIG antibody in plasmids. Phages are formed by recombination which express heavy and / or light chains in the tail region of MIG. Using the method of Example II, this phage preferably grows in bacteria that are resistant to the parental phage and selected because they have the structure to be identified, MIG, on the surface.

This structure is expressed on the pilus as shown in Example III or on other surface proteins used as receptors in the infection.

Example VI-B

^ E_. coli bacteria containing L-chain V genes of anti-MIG antibodies in plasmids are prepared according to standard methods. Phages are grown in these bacteria to obtain recombinant DNA. In some phages, the V or L genes are expressed in the cilia, but JL? Π

this is incompatible with survival. The same method is used for heavy chain variable genes. To eliminate the parental phages, antibodies specific for cilia are made, and all phages that have normal cilia are immunized with their host bacteria J by absorption and precipitation. Rare phages remain, which are either imperfect or have Z or L chains. They are concentrated by ultracentrifugation and used to infect bacteria expressing MIG, or MIG-like structures, on their surface, as described above. Attracted via non-covalent forces, the phages expressing Ig genes infect their bacteria. The resulting progeny are "hybrid" phages, phage pairs that continue to cause co-infection. The specificity of this hybrid for MIG

8520062-23 is determined as described above. The phage are visualized as in Example I.

Example VII

Carbohydrates, either as mono-, oligo- or polysaccharides, are coupled to bacteriophages, which are then rendered fluorescent or radioactive or coupled to an enzyme. These samples are used to identify lectins in solution, using a fluorometer, on solid phase or on cells. The phages are visualized as described in Example I.

Example VIII

Lectins are coupled to bacteriophage, which is visualized as in example I. This conjugate phase is used to determine carbohydrates for which the lectins are specific, either in solution, as in example VII, or cells, or on solid phases.

2o Preferred embodiment

In the preparation of bacteriophage mutants for the recognition of mouse immunoglobulin (igG),

Escherichia coli bacteria prepared in Petri dishes and irradiated with UV light to kill 50-90% of the bacteria. A suspension of T4 bacteriophages is also treated with UV light to kill about 80% of the bacteriophages and cause mutations. Bacteria are infected with the bacteriophage, and the bacteriophage is grown, collected and treated with chloroform. The phage suspension is then concentrated, by precipitation with polyethylene glycol or by ultracentrifugation, to obtain a suspension of more than 12 ..

than 10 infectious units / ml.

The bottom of a plastic Petri dish is coated with mouse IgG (MIG) directly by incubation at 25 ° C and pH 9.2 overnight, or by using poly-L-lycin as a coupling agent. This MIG has no anti-phage antibody activity, ie it is either monoclonal for another specificity, or it is pre-absorbed with phage or only it; FC part is used. The phage is added to the Petri dish and incubated at 37 ° C for 1 hour. The plate is carefully washed to remove any unbound phage present.

The treated and washed plate is then used to grow the phage directly by adding bacteria in soft agar. The plate is first repeatedly washed to remove the non-specifically bound phage.

15

To increase the chance of acquiring head mutations that survive, the phage are grown at a higher temperature (42 ° C) and the selection process is repeated 2-3 times.

As a preliminary study for phage clones that recognize MIG, plates are coated with MIG and the parent strain of the phage is added to several plates at the same time as the mutants, incubated, washed, and finally bacteria are added.

Although the parent strain gives few spots, the mutants that bind to MIG give many spots and even confluent lysis of bacteria.

25

In a related method, phage colonies are picked up on an adsorbent paper, and made

fluorescent MIG added, incubated to bind MIG

to promote the mutant, washed and assessed with a UV light scanner. The affected mutants are retrieved and cloned in the original gel. The phages are collected, treated with chloroform, collected by precipitation with polyethylene glycol or by ultracentrifugation, and added to micropiglets coated with MIG. After incubation at 37 ° C for one hour, the fluorescence is determined and the phage clone exhibiting the greatest fluorescence is then grown and retested for its ability to bind MIG in the same system as before.

:

The bacteriophage that shows binding to MIG, i: 5 is visualized by coupling with fluorescein isothiocyanate.

i

To test the value as a phage reagent, human lymphocytes are treated with MIG anti-human T-cell monoclonal antibodies. The cells are washed and the fluorescent phage added. For control, the fluorescent phage is added to human lymphocytes that have not been treated with the monoclonal antibody. After treatment for 30 minutes, the lymphocytes are washed and the cells are examined under the microscope with UV light. The intense fluorescent 15 'staining can indicate 80-90% of human peripheral blood lymph nodes by phage recognition of mouse IgG on the lymphocyte surface.

Technical applicability

The method according to the invention is particularly suitable for obtaining an effective clinical trial method and for a research package comprising effective amounts of a selected bacteriophage coupled with a facial hair-making agent. Such a package is inexpensive; usable in the hands of an appropriately trained clinical laboratory assistant; and very suitable for clinical use, with many tests usually carried out in a relatively short period of time.

; 30 8520062

Claims (22)

1. Method of identification and quantification; ringing of molecular and cellular materials present in a test sample, in which method one successively: (a) selects a bacteriophage which has been adapted by genetic or chemical manipulation to bind to said molecular or cellular materials; (b) couples to the selected bacteriophage a visualizing agent, or analyzing agent, with a measurable signal, which analyzing agent is from the group of fluorescent agents, radioactive isotopes, enzymes, metals and staining agents; (c) combining the molecular or cellular materials in the test sample with the bacteriophage coupled in step (b) to obtain a conjugate phase in the test sample; and | (d) subject the conjugate phase to analysis, i taking the measurable signal from the coupled analyte | | used. i,,, Improved method for the identification and quantification of molecular and cellular materials present in a test sample, wherein one successively: ^ (a) selects a bacteriophage characterized by the ability to recognize said molecular and cellular materials; (b) incorporates into the selected bacteriophage a visualizing agent selected from the group of fluorescent agents, radioactive isotopes, enzymes, metals and colorants; (c) combining the test sample with the selected bacteriophage under binding conditions to obtain a conjugate phase in the test sample, which conjugate comprises the selected bacteriophage and the visualizing agent incorporated therein together with the molecular and / or cellular material; and (d) subjecting the conjugate phase to analysis using the visualizing agent. A method according to claim 1, wherein the β: 120 bacteriophage is selected for the ability to head bind to molecular or cellular materials and wherein said! selection is effected to obtain a conjugate phase, in which method one further sequentially: (a) mixes the bacteriophage with bacteria; (B) irradiating the mixture with ultraviolet light to effect mutation of the bacteriophage; (c) the mutant bacteriophage multiplies in the presence of bacteria in i 8520062 i% - 31 - I to obtain a population of i. i | the mutant bacteriophage; | (d) purify the population of the mutant bacteriophage; ί i- (e) the purified mutant bacteriophage binds to a cell surface or to a molecular material; and (f) multiplying the bound mutant bacteriophage in bacteria j. The method of claim 1, wherein the bacteriophage is selected for the ability to bind through the head to molecular or cellular materials and wherein said binding is effected to obtain a conjugate phase by successively: (a) mixing the bacteriophage with a substantial excess of bacteria; (b) irradiating the mixture with ultraviolet light to effect mutation of the bacteriophage; (c) culture mutant bacteriophage in the presence of bacteria; (d) purify the cultured mutant bacteriophage; 8520062; - π- j (e) the purified mutant bacteriophage binds to a cell surface or a molecular material; and i (f) culture the bound mutant bacteriophage. j The method of claim 2, wherein the \ 1Γ1 molecular material comprises immunoglobulins. ! IU The method of claim 2, wherein! : 5 the molecular material comprises immunoglobulins. The method of claim 2, wherein the molecular material is coupled to a solid phase. The method of claim 2, wherein the molecular material is coupled to a solid phase. The method of claim 2, wherein the; cellular material surface glycoproteins of animal, ^ cells. The method of claim 2, wherein the cellular material comprises animal cell surface glycoproteins. The method of claim 1, wherein the bacteriophage is selected for the ability to bind via the tail to molecular or cellular materials and wherein said selection is triggered to obtain a | 20 conjugate phase, in which process one successively: (a) injecting the molecular or cellular material into an animal host for the preparation of antibodies therefor; (b) the counter said molecular or cellular | Recover and purify material specific antibodies; I (c) couples the purified specific antibodies to II a solid phase; (d) treats the coupled antibodies with a significant excess of bacteriophage resistant bacteria,! 2q whereby some mutant bacteria bind to the antibodies; | (e) the mutant bacteria selected by their ability to bind to said antibodies, breeds and! collects; | 8520062 * ▼ (f) irradiates the selected bacteriophage with ultraviolet j! light; | (g). separately a bacteriophage-sensitive bacterium! j! trunk irradiates with ultraviolet light; (H) mixing the irradiated bacteriophage from step (f) with the irradiated bacteria from step (g); (i) approximately equal portions of the products of steps (e) and (h) mixing under spot development conditions, | whereby mutant bacteriophages are formed and give clear spots; eri I 10 (j) wins the mutant bacteriophage from the bright spots and this! in the bacteria of step (e) multiplies, selecting a mutant bacteriophage which can bind to said molecular or cellular materials. The method of claim 6, wherein the molecular material comprises immunoglobulins. The method of claim 1, wherein the bacteriophage is selected for its ability to bind via the tail to molecular or cellular materials and wherein said binding is effected to obtain a conjugate phase by successively: (a) molecular or cellular material into an animal host for the preparation of anti-! bodies therefor; ! (b) wins the antibodies. and purifies; | (C) couples the purified antibodies to a! solid phase; | (d) treating the coupled antibodies with a significant excess of bacteria, which bacteria are chosen because of their resistance to the selected bacteriophage, whereby the bacteria bind to the antibodies; (e) I selected by said antibodies; grow and collect bacteria; (f) irradiates the selected bacteriophage with | ultraviolet light; | 30 (g) separately irradiates a bacterial strain sensitive to the selected bacteriophage with ultraviolet | light; j (h) mix the irradiated bacteriophage of step (f)! with the irradiated bacteria of step (g); 85 2 0 0 6 2 ................................ - 28- (i) approximately equal portions of the products of steps (e) and (h) mixing under culture conditions to form mutant bacteriophages, whereby tail binding of the mutant bacteriophage and the antibody selected j; c i • gives bacteria a conjugate phase; and j (j) that portion of the j grown in step (i); The conjugate phase wins that the antibody-selected bacteria can lyse: j of step (e), thereby selecting a conjugate phase which comprises mutant bacteriophage comprising said molecular | and / or bind cellular materials. j The method of claim 6, wherein the! molecular material includes immunoglobulins. ; The method of claim 1, wherein the cellular material comprises bacteria. The method of claim 1, wherein the material to be identified comprises bacteria. ! IC The method of claim 8, wherein the surface of the bacteria is coated with receptors controlled by plasmid fci. The method of claim 8, wherein the identifiable material comprises bacteria the surface of which is covered with plasmid-controlled antigens. The method of claim 1, wherein the analyzing agent of step (b) is coupled to the selected bacteriophage after the combination with the test sample to obtain a conjugate phase; as in step (c). 11. The method of claim 1, wherein the selected bacteriophage is chemically coated with antibodies to the material to be identified. The method of claim 1, wherein the visualizing agent is incorporated into the selected 20 ... bacteriophage after binding to the test sample. : The method of claim 1, wherein the selected bacteriophage is chemically coated with antibodies; against the material to be identified. 12. The method of claim 1, wherein the selected bacteriophage is linked to the material to be identified via a hybrid antibody. The method of claim 1, wherein the: 2S. .... . selected bacteriophage is linked to the material to be identified via a hybrid antibody. The method of claim 1, wherein the selected bacteriophage is coated with carbohydrates, whereby the conjugate phase serves to identify and quantify lectins. 13. Method according to claim 1, wherein the selected bacteriophage is coated with carbohydrates, whereby the conjugate phase serves for the identification and quantification | of lectins. ! The method of claim 1, wherein the j ______________________________...................................._ ___ ______________________________ i 1520062 * * * 33. I selected bacteriophage is coated with lectins, whereby | j the conjugate phase serves to identify and quantify j j carbohydrates. ; i j The method of claim 1, wherein the j; selected bacteriophage is coated with lectins, whereby the I I j I i 8520062 · - · - i i; ; i ί ___________________________-............................................... .................................................. .................................................. .............................. J; - 29- I conjugate phase serves to identify and quantify; carbohydrates. The method of claim 1, wherein the; I 5 analyzing agent is a fluorescent agent. | i | The method of claim 1, wherein the visualizing agent is a fluorescent agent. | The method of claim 1, wherein the analyzer is a radioactive isotope. The method of claim 1, wherein the visualizing agent is a radioactive isotope. j The method of claim 1, wherein the analyzing agent is an enzyme. I The method of claim 1, wherein the visualizing agent is an enzyme. j The method of claim 1, wherein the analyzer is a metal. The method of claim 1, wherein the visualizing agent is a metal. iq. The method of claim 1, wherein the visualizing agent is a coloring agent. The method of claim 1, wherein the cellular material of the test sample is pathogens in pathalo-; gic liquids, and the visualizing agent is a fluorescent or an enzyme. The method of claim 20, wherein the! selected bacteriophage is coated with antibodies specific for the pathogens. 22. Package for the identification of bacteria, eucariotic cells and other molecular materials, which package contains measured, effective portions of selected bacteriophage coupled with a visualizing agent. i! j j i j; 8520062 I-30-I i; I j i - AMENDED CONCLUSIONS -! ! i (received by the International Bureau on August 12, 1985 j (12.08.85); original claims 1,2,6,8-10,15-20 and 22 amended; other conclusions unchanged) The method of claim 1, wherein the analyzing agent is a coloring agent. The method of claim 1, wherein the; ^ cellular material of the test sample includes pathogens in pathological fluids, and the analyzer is a fluorescent or an enzyme. ; 21. The method of claim 20, wherein the selected bacteriophage is coated with antibodies specific for the pathogens. : 22. Package for the identification of bacteria, eucariotic cells and other molecular materials, which package of measured, effective portions of selected bacteriophage,! linked to an analyzer. 25 t; i: j; | . i l ..... ...... __ _________________ ____ i 8520062