Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/586—Liposomes, microcapsules or cells

Definitions

• This invention relates to the field of immunology, providing a novel and reliable method for the identificatio and assay of materials of both procaryotic and eucaryoti ⁇ ⁇ c cells, and comprises a selected bacteriophage coupled with visibility agent. There is further provided an assay kit for the ready determination of bacteria, eucaryotic cells, and various molecular materials.
• antibodies are used almost exclusively to identify molecular and cellular structures that do not reveal themselves otherwise. For example, they are used to distinguish one protein from another, one poly-
• the interaction between the antibodies and the "identified" structure may be measured by a variety of methods aimed at making visible the presence of the antibodies.
• an antibody bound to the structure, cell, or solid phase to be identified is directly made
• a second antibody is made visible and is directed against the first antibody.
• protein A of Staphylococcus aureus, Cowan I strain is made visible and it binds to the first antibody.
• Another class choir - of reagents used to make the antibody visible is represented by biotin and avidin which recognize each other and can be used in conjunction with antibodies. Fluorescent material, enzymes, radioactive materials, large particles, such as erythrocytes, or beads have been used to make the antibody visible directly or indirectly. Each of these procedures has shortcomings and limitations which may be illustrated by the use of fluores ⁇ cent materials to visualize the antibody. If the amount of antibody bound by a cell is too small the fluorescent reagents fail to detect it.
• bacteriophages have been con- ' sidered either too small to serve as efficient carriers or to be sticky in a nonspecific way, particularly due to their tail fibers.
• the only use that bacteriophages have had in identification experiments has been to use them as viruses for their bacterial host or to put antigens on their sur ⁇ face, usually through a mild chemical process of coupling, and then to use antibodies against these antigens to block the ability of the bacteriophage to infect bacteria in bacteriophage neutralization systems.
• bacteriophage was used only as living virus in Haimovich, et al. , U.S. patent no. 3,171,705 and in Young, U.S. patent no.
• Bacteriophages which are bacterial viruses, have not been considered in this category for these obvious reasons. First, they are not used for any hemagglutination or hemagglutination inhibition assays; these are based on characteristics of only certain animal viruses. Second, it is well known that bacteriophages have specialized struc ⁇ tures, most with the appearance of a tail, that are used for recognition of the receptor on bacteria, leading to infec ⁇ tion. It is well known that once this " tail binds to its receptor, even when it is not on bacteria but in solution, the nucleic acid is evacuated and the virus becomes noninfec tious.
• This invention relates to a novel method for the identification and quantification of molecular and cellular materials of both procaryotic and eucaryotic cells wherein a test sample is combined with a selected bacteriophage under binding conditions to provide in the test sample a conjugate phase, comprising bacteriophage coupled with the molecule or cells sought to be identified.
• a visibility agent is incor ⁇ porated in the bacteriophage, either before or after the binding step, to improve greatly the recognition of the test material in conventional analytical assay techniques.
• the bacteriophage may be selected to bind through its head or through its tail. In the latter instance, this is accomplished by "external imaging" whereby the bacteriophage is modified to perform i the manner of an antibody.
• mutants of the bacteriophage are employed.
• the method of this invention can be adapted to the analysis of proteins, carbohydrates, leetins, bacteria of various types, pathogens, and miscellaneous molecular or cellular materials present in tissues, cells or fluids.
• test material in a form suitable for use in any selected analytical instrument
• the recognition of a structure by an agent of ide tification such as an antibody can be subdivided into two parts, the "intelligence", i.e., the specific site that binds a complementary structure and the “visualizer”, i.e., the structure that is somehow made visible.
• the "intelligence” is provided by the combining site of the antibody molecule and the “visualizer” by the rest of the molecule with its molecular attachments (fluorescent, radio ⁇ active, etc.).
• the antibodies are coupled to bacterio ⁇ phages.
• This coupling may be covalent, as with glutaral- dehyde or bifunctional reagents, or noncovalent, as with hybrid antibodies.
• the bacteriophages can be selected and constructed to provide the "intelligence", i.e., to function as antibodies, and also to carry the "visualizer”.
• bacteriophages are made visible and are either coated with antibodies or binded naturally through their receptor.
• • bacteriophages are used as the visualizer, or carrier of intelligence, which is provided by the antibody.
• Antibodies may be coupled to bacteriophages chemically, employing bi- functional reagents, such as glutaraldehyde or other covalent or non-covalent coupling agents.
• Bacteriophages are coated with avidin or biotin to bind to the antibody that has biotin or avidin, respectively. The binding is achieved through biotin-avidin recognition.
• Bacteriophages serve as visualizers by linking to the structure to be identified with the help of hybrid anti ⁇ bodies. These are directed with one combining site against the bacteriophage and with the other against a first anti ⁇ body or the structure to be identified.
• the bacteriophage can also be coupled to lectins or carbohydrates for recogni ⁇ tion of the respective complementary structures.
• the visualizer provided by the bacteriophage is obtained with the help of fluorescent dyes, other dyes, radioactive isotopic material, enzymes, or metals such as silver or gold.
• the bacteriophage may also be * engineered to contain the enzyme of use.
• Bacteriophages can be selected to provide both intelligence and visualizer through genetic manipulation, providing them with a "combining site". After mutation, the bacteriophages are then selected for the property of the head to bind to molecules such as immunoglobulins or to glycoproteins or proteins of animal cells.
• the mutants are obtained, for example, by ultraviolet light irradiation of both bacteriophage and bacteria, followed by the growth of the bacteriophage.
• the bacteriophage is harvested, purified and the selection pressure_is applied; namely, binding to cell surfaces or to molecules coupled to a solid phase.
• the bacteriophage is made fluorescent for cell surface identifi ⁇ cation directly or after the cell has been treated with the antibody recognized by the bacteriophage.
• the lethal nature of head mutations is avoided by using very large numbers of particles and by selecting temperature-resistant mutants. thus selecting for very rare events that are still com ⁇ patible with bacteriophage survival.
• mutants can be prepared by using the tail's ability to bind to a specific structure on the surface of bacteria.
• mutation and selection bacterio ⁇ phages are selected which have their tail capable of recog ⁇ nizing particular structures. This is done by "external imaging", an unobvious analogy with internal imaging in the antiidiotype network of antibodies, as discussed by Urbain, et al.. Progress in Immunology, 1980, Academic Press, Vol. I, pp. 81-92.
• the molecule to be identified, "X” is injected in 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.
• bacteria are selected for resistance to bacteriophage "Y” from bacteriophage-sensitive parental strain. These bacteriophage-resistant bacteria lack recep ⁇ tors for the bacteriophage. Bacteria that bind to these antibodies are selected and grown so that they will have a structure that mimics "X". The selection of bacteria capable of expressing "X"-like structures can be verified by their ability to bind to purified anti "X” on the bacteria. It is most unexpected that such a structure can be the receptor for the bacteriophage. These bacteria are then mixed, in approximately equal parts, with the bacteriophage- sensitive bacteria which had been UV-irradiated and treated with mutagenized (e.g., UV-irradiated) bacteriophage.
• mutagenized e.g., UV-irradiated
• the mutant bacteriophages emerging from sensitive bacteria which are capable of recognizing the receptor on the bacteriophage-resistant bacteria, will grow in these bacteria.
• all confluent lysis will be turbid since only the bacteriophage-sensitive bacteria are lysed, except for a few clear plaques in which both bacteria will be lysed as a result of the mutant.
• the plaques are removed to an absorbent material and the mutants that recognize "X" are detected with an "X" probe which is made visible, i.e.. radioactive, fluorescent, etc.
• the mutant bacteriophages are traced on the plate and cloned on their new bacterial host.
• the bacteriophages are made to recognize a parti ⁇ cular antigen by other genetic manipulations. Bacteria are used that have some surface structures controlled by plas ids. For example, the gene for "X” is incorporated in the plasmid. Bacteria which display "X" on their surface are then selected as described above. These surface proteins, or glycoproteins are on structures used by the bacteriophage as receptors. By selecting bacteriophages that recognize these structures, as their infection receptors, bacteriophages are obtained that recognize "X". Accordingly, an external image of the antigen is obtained on bacteria.
• bacteriophage tail have the same sequences as heavy and/or light chains of im unoglobulin, through recombination with immunoglobulin genes in plasmids.
• Parental bacteriophages with the normal tail sequences are removed by absorption with natural hosts. These are mutated and selected for recognition of "X" through the methods described above.
• a complete heavy and light chain arrangement is obtained by coinfecting bacteria having "X" on the surface with two bacteriophages, one expressing Vg gene products and the other V j . gene products.
• one means involves assembling a kit, comprising an appro ⁇ priate bacteriophage which is coupled with a visibility agent, for use in the identification of bacteria, eucaryotic cells, and other molecular materials. A ⁇ number of portions of the phage can be afforded, each in an amount selected to be effective in the contemplated assay. '
• Bacteriophages are coupled to antibodies, mono ⁇ clonal or polyclonal, after having been made fluorescent, radioactive, or coupled to peroxidase, and are used as a staining, or identification, agent.
• a coupling agent e.g., glutaraldehyde or other bifunctional reagent.
• the phage is made fluores ⁇ cent by coupling with fluorescein isothicyanate, 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 radio-active either by incorporation of radioactive materials in its protein or nucleic acid or by conventional procedures of coupling radioactive materials to proteins.
• a metal such as silver is added to the phage by conven tional procedures.
• 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 in tryptic soy broth (DIFCO) and mixed with phage for a multiplicity of infections of 0.5 to 1. The 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, the top soft agar layer was collected and treated with chloroform and' with 0.01M EDTA to precipitate the non-phage material.
• DIFCO tryptic soy broth
• the mixture was centrifuged at 5000 G for 10 minutes and the supernatant was collected. To remove free nucleic acids, the supernatant was treated with 20 "-* ug/ml. of DNAase and 20 ug/ml. of RNAase at 37°C. for 1 hour. To wash the phage, NaCl and polyethylene glycol (PEG) were added to final concentrations of 0.5 M and 6%, respectively, and the mixture was incubated at 4°C. for 18 hours.
• PEG polyethylene glycol
• the phage in suspension in 0.1 M phosphate buffer at pH 8.5, was mixed with glutaraldehyde to a concentration of 1% and incubated at 25°C. for 1 hour. Excess glutaraldehyde was removed by precipitating the phage with 0.5 M NaCl and 6% PEG and re-suspending in buffer at pH 8.5.
• Antibody, 0 anti rabbit Ig was added to obtain 1 g. antibody/1 mg. phage protein. After 2 hours incubation at 4°C. with rabbit lymphocytes and the cells were washed three times by centri ⁇ fugation at 900 G for 5 minutes.
• the binding of the fluorescent phage was compared 5 with that of fluorescent phage coated with either normal IgG instead of antibodies or with fluorescent anti Ig antibodies.
• the cells were examined under the fluorescence microscope.
• the cells with macrophage character i.e., large
• the cell preparation treated with anti-Ig antibody-coated phage accounted for 47% of the cells with surface fluorescence while those treated with normal Ig-coated phage had only 1-2%.
• the surface fluorescence was made more intense 5 than that of the same cells treated with anti-Ig fluorescent antibodies.
• the anti-Ig antibody-coated phages showed specificity of binding and delivered intense fluorescence.
• Example I-B Concentrated, purified bacteriophage is treated with a heterobifunctional reagent. Rabbit anti-allotype antibody (anti b_j) is thiolated and a bridge between the two is formed so that the rabbit antibody becomes coupled to the phage. As a control, the phage is then made fluorescent. The phage-antibody complex is then mixed with the cells, washed, and is examined, either with a microscope or other appropriate instrument. The degree of fluorescence is regulated through the degree of use of amino groups on phage proteins.
• Example I-C The phage, coated with antibody, is employed to treat fixed tissue sections. A section of lymph node is treated with a fluorescent phage preparation, washed, and examined under UV light. In an alternate procedure, coated phage is revealed by final addition of the substrate according to standard methods. This method is also used for phages selected to bind spontaneously.
• Example I-D The phage, selected to recognize MIG either by the tail or by the head, is mixed with a solution containing MIG antibodies, e.g., monoclonal antibodies. This phage is either fluorescent or radioactive. After washing by precipi ⁇ tation with PEG, it is used to replace phages coupled chemically with antibody. The phages are made visible by an appropriate coupling procedure.
• MIG antibodies e.g., monoclonal antibodies.
• Bacteriophages are designed to be used in rapid diagnosis of pathologic materials containing bacteria. Joint fluid or exudate is smeared, fixed, phages are added, and their presence visualized under proper instrumentation, such as UV light for fluorescence, bright field for enzymes, etc. By using automated computerized scanners the process is made very rapid. The binding of phages due to antiphage antibodies is avoided by competitive saturation with free phage protein or by treating the sample with formaldehyde. Very small numbers of bacteria, even if they are dead, are readily identified. Two-step addition of complex phage sus ⁇ pensions against a variety of possible pathogens, followed by individual suspensions in those products that appear positive, permits a rapid identification of rare micro ⁇ organisms.
• T4 bacteriophages were grown and purified as in Example I-A.
• Various amounts of fluorescein isothiocyanate (FITC) ranging from 0.5 mg. to 8 mg., were added per 1 mg. of phage protein.
• the phage suspension was adjusted to pH 9.3 with 0.1 M Na2C ⁇ 3.
• FITC was added with stirring at 4°C, stirring continued for 2 hours and then incubated at 4°C. for an additional 20 hours. Excess FITC was removed by dialyzing the suspension against 0.1 M phosphate buffer (pH 8.5) at 4°C. for 3 days.
• 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.
• E. coli USC 106 which is a phage-resistant mutant of YS57, Bacillus globigii and Salmonella schottmulleri were used.
• phages were mixed with formaldehyde-fixed bacteria at 25°C. for 10 minutes and then washed 3 times to remove the unbound phage.
• the phage sensitive E. coli YS57 became intensely fluorescent so that even one microorganism could be clearly seen under the fluorescence microscope.
• Phage prepara ⁇ tions (1), (2) and (3) i.e., up to an FITC/protein molar ratio of 11.3, were made intensely fluorescent, while the mutant USC 106, B. Globigii and S . schottmulleri.
• the phage specificity was maintained at a molar ratio of 11.3, which is similar to that routinely used for antibodies.
• the phage particles have about 500 times more protein than do antibodies, the phage particles also provide that much more total fluorescence.
• Example III-A To obtain mutants of phages that recognize mole ⁇ cules and cells, the first condition is that the phage should not bind naturally to such cells or molecules.
• phages were prepared as in Example I-A and made fluorescent to a FITC/phage protein molar ratio 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 mononuclear cells were purified from peripheral blood by the Ficoll-hypaque method, washed, and incubated at 4°C. for 1 hour with monoclonal mouse anti-human T cells, and washed again.
• Rabbit lymphoid cells were obtained from spleen and lymph nodes by routine procedures, mixed and washed. Suspensions containing 10> cells/ml. (human or mouse) were treated with 10 12 fluorescent phage particles. prepared as in Example I-A but not coated with any anti ⁇ bodies. After incubation at 4°C. for 1. hour, the cells were washed and examined under the microscope. No surface fluorescence was observed, indicating that T4 bacterio- ) phages, made fluorescent, do not stick non-specifically to either MIG, human lymphocytes, or rabbit lymphocytes. This surprising observation provided the basis for selecting for bacteriophages that bind.
• bacteriophages that bind to struc tures other than those that they recognize naturally is done in two ways: by screening different existing bacteriophages from various collections and by synthetic manipulations.
• An example of screening is the following.
• Bacteriophage T4 does not bind to fresh suspen ⁇ sions of human or rabbit lymphocytes.
• smears were prepared of rabbit and human blood cells and were treated with formaldehyde. The smears were then treated for 30 minutes with a suspension containing 10-1 4 phage particles per ml. which were made fluorescent by.treat ment with FITC. The smears were washed, a cover slip placed on top, and examined under a fluorescent microscope. A very intense fluorescence was seen only in the cytoplasm of the leukocytes. The nucleus, the membrane, and the red blood cells were not fluorescent. This test shows that, somehow, through an unknown and unobvious mechanism, some structure in the cytoplasm of the white cells is recognized by the FITC-labeled T4 phage.
• the selection of bacteriophages is also done through synthetic manipulations.
• Escherichia coli, strain YS57 are prepared in petri dishes, as in Example I-A, and are irradiated with UV light to obtain 50-90% killing of the bacteria; this step is done to trigger 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, harvested and treated with chloroform. The phage suspension is then concentrated by precipitation with poly ⁇ ethylene gly ⁇ ol or by ultracentrifugation to obtain a sus ⁇ pension of over 10 12 infective units/ml.
• mouse IgG mouse IgG
• 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.
• MIG mouse IgG
• This MIG does not have antiphage antibody activity, i.e., it is either monoclonal for another specificity or it is pre- absorbed with phage or only the Fc portion is used.
• the phage is added to the dish and is incubated at 37°C. for 1 hour. The plate is carefully washed to remove any unbound phage.
• mouse IgG is attached to particles, erythrocytes, bacteria or beads, either chemically or through its antibody function.
• the plate that is treated with phage and washed is used to grow the phage directly by adding bacteria in soft agar.
• the plate is first washed repeatedly to remove the phage that is not bound speci ically.
• the phage is also grown at higher temperatures, e.g., 42°C.
• the phage colonies are picked up and grown in susceptible bacteria and re ⁇ loned 2-3 times.
• Example III-B In a companion method to that, of Example III-A, the phage colonies are picked up on an adsorbent paper. Either radioactive or fluorescent MIG is added, incubated to promote binding of MIG to the mutant, is washed and then examined, respectively, by a scanner for radioactivity or with UV light.
• the relevant mutants are retraced to the original gel and cloned. To obtain additional evidence all colonies formed are collected and grown in bacteria, each clone in two tubes, one containing amino acids labelled with l ⁇ C.
• the phages are collected, treated with chloroform, collected by precipitation with polyethylene glycol or by ultracentrifugation, and are added to microwells coated with MIG. After incubation at 37°C. for one hour the wells are washed, sodium dodecylsulfate (SDS) solution is added to remove bound radioactive material and its c.p.m. determined.
• SDS sodium dodecylsulfate
• Example III-C The bacteriophage that demonstrates binding to MIG is made visible by any of the usual procedures. It is made fluorescent by coupling with fluorescein isothiocyanate, rhodamine or other fluorescent dyes, by treatment with ethidiu bromide, which binds to the phage DNA, or by other dyes that bind to nucleic acids.
• the phage is made radio ⁇ active either by incorporation of radioactive materials in its protein or nucleic acid or by conventional procedures of coupling radioactive materials to proteins. In the alterna ⁇ tive, a metal such as silver is added to the phage by conven tional procedures.
• human lymphocytes are treated with MIG anti-human T cell ono- ⁇ lonal antibodies.
• the cells are washed and, for example, the fluorescent phage is added.
• cent phage is added to human lymphocytes not treated with the monoclonal antibody.
• the intense fluorescent staining of the 80-90% of the human peripheral blood lymphocytes demonstrates that the phage recognizes mouse IgG on the lymphocyte surface.
• the lack of fluorescence of cells treated with parental phage instead of the mutant, or pre ⁇ treated with normal MIG instead of anti T cell antibody, offers a control.
• purified human B cells are shown not to become fluorescent when treated like the unseparated peripheral blood lymphocytes.
• Example IV The selection of mutants by external imaging requires the preparation of bacteria that have phage receptors which mimic a certain macromole ⁇ ule, e.g., mouse IgG (MIG), to be identified.
• MIG mouse IgG
• MIG is injected into rabbits or rats to induce anti MIG antibodies.
• the following procedure is designed to make a bacteriophage work just like an antibody by using its tail as a combining site.
• the anti MIG antibody is purified by affinity chromatography and is coupled to a solid phase, such as the bottom of a petri dish, by using poly-L-lysine.
• E. coli which is resistant to the phage, is grown, washed, suspended in physiologic buffered saline, added to the petri dish and is incubated at 4°C. for one hour. The petri dish is care ⁇ fully 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 bacteria are treated with mutagens to increase the number of mutants.
• Bacteria that are selected are cloned and each clone is tested for binding by the anti-MIG antibody by con ⁇ ventional methods. Bacterial mutants grow, when allowed to attach to the bottom of plates coated with the antibody against MIG, to a greater extent than the parental strain, thus providing a selective advantage to the mutant.
• the phage sensitive bacteria are UV 5 irradiated, the phage is UV irradiated and is then mixed with phage-sensitive bacteria for infection and generation of mutants.
• the phages and bacteria can be first mixed and then UV irradiated. These bacteria are mixed in equal proportions with phage-resistant bacteria that are
• the phage lyses all phage-sensitive bacteria but does not affect the phage- resistant ones. When a mutant phage appears, which recog ⁇ nizes the MIG-like structure on the phage-resistant bacteria, a clear plaque is formed.
• Example V Some surface receptors, or antigens, on bacteria are controlled by plasmids.
• An example is the sex pilus on E. coli. This pilus is then recognized by sex-specific JJ phages, such as 0X174.
• the gene for the constant domain of either the heavy chain or the light chain of MIG is inserted into ' the plasmid that controls the sex pilus.
• Bacteria that express MIG in place of or together with pilus proteins are selected by using a solid phase with anti-MIG antibodies. Phages recognizing this pilus are then selected and shown to recognize MIG as before.
• the phage resis ⁇ tant bacteria are selected by the positive pressure of the anti MIG antibody. The phage is visualized as in Example I.
• Example VI-A Bacteriophages are grown in bacteria that have variable genes of an antibody directed against MIG in plasmids. By recombination, phages are generated that express in the tail region of MIG heavy and/or light chains. By using the method of Example II, this phage grows preferen ⁇ tially in bacteria resistant to parental phage that have been selected to have on the surface the structure to be identified, i.e., MIG. This structure is expressed on the pilus as shown in Example III, or on other surface proteins that are used as receptors in infection.
• Example VI-B E. coli containing L chain V genes of anti-MIG antibodies in plasmids are prepared according to standard procedures. Phages are grown in these bacteria to obtain recombinant DNA. Some phages have the V j * . or L genes expressed in the tail fiber but this event is incompatible with survival. The same procedure is applied for variable genes of heavy chains. To eliminate the parental phages, antibodies are made to be specific for tail fibers and all phages having expressed normal fibers are eliminated by absorption and precipitation with their host bacteria. Rare phages remain which are either defective or have H or L chains expressed. They are concentrated by ultracentrifu- gation and used to infect bacteria that express MIG, or MIG-like structures, on their surfaces as described above.
• the phages expressing Ig genes infect their bacteria.
• the resulting progeny are "hybrid" phages, pairs of phages that will continue to cause coinfe ⁇ tion.
• the specificity of this hybrid for MIG is determined as described above. The phage is visualized as in Example I.
• Example VII Carbohydrates, either as mono, oligo, or poly- saccharides, are coupled to bacteriophage which is then made fluorescent, radioactive or coupled to an enzyme. These probes are used to identify lectins in solution, with the aid of 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 made visible as in Example I. . This conjugate phase is used to probe for carbohydrates for which the lectins are specific, either in solution, as in Example VII, on cells, or on solid phases.
• Escherichia coli In the preparation of bacteriophage mutants to recognize mouse immunoglobulin (IgG), Escherichia coli, are prepared in petri dishes and are irradiated with UV light to obtain 50-90% killing of the bacteria. 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, harvested, and treated with chloroform. The phage suspension is then concentrated, by precipitation with poly ⁇ ethylene gly ⁇ ol or by ultracentrifugation, to obtain a sus ⁇ pension of over 1012 infective units/ml.
• IgG mouse immunoglobulin
• MIG mouse IgG
• 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.
• MIG mouse IgG
• This MIG does not have antiphage antibody activity, i.e., it is either monoclonal for another specificity or it is pre- absorbed with phage or only the Fc portion is used.
• the phage is added to the dish and is incubated at 37°C. for 1 hour. The plate is carefully washed to remove any unbound phage.
• the treated and washed plate is then used to grow the phage directly by adding bacteria in soft agar.
• the plate is first washed repeatedly to remove the phage that is not bound specifically.
• the phage is grown at a higher temperature (42°C.) and the process of selection is repeated 2-3 times.
• phage colonies are picked up on an adsorbent paper, and fluorescent MIG is added, incubated to promote binding of MIG to the mutant, washed, and examined by a scanner with UV light.
• the relevant mutants are retraced to the original gel and cloned.
• the phages are collected, treated with chloroform, collected by precipitation with polyethylene glycol or by ultracentrifu- gation, and are added to microwells coated with MIG. After incubation at 37°C. for one hour the degree of fluorescence is determined and the phage clone that exhibits the highest fluorescence is then grown and retested for its ability to bind to MIG in the same system as above.
• the bacteriophage that demonstrates binding to MIG is made visible by coupling with fluorescein isothiocyanate.
• human lymphocytes are treated with MIG anti-human T-cell mono ⁇ clonal antibodies. The cells are washed and the fluorescent phage is added. As a control the fluorescent phage is added to human lymphocytes not treated with the monoclonal anti ⁇ body. After treating for 30 minutes, ⁇ the lymphocytes are washed and cells are examined under the microscope with UV light. The intense fluorescent staining of the 80-90% of the human peripheral blood lymphocytes indicates recognition by the phage of mouse IgG on the lymphocyte surface.
• This inventive method lends itself most especially to the affordance of an effective clinical test procedure and to an assay kit comprising effective portions of a selected bacteriophage, coupled with a visibility agent.
• a kit is inexpensive; operable in the hands of a suitably trained clinical laboratory assistant; and most suitable for clinical use where many tests are customarily conducted in a relatively short period of time.

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• 1985
  • 1985-03-15 GB GB08527324A patent/GB2181542A/en not_active Withdrawn
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