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EP1453959A2 - Procede de selection et d'identification de molecules peptidiques ou proteiques par presentation a la surface de phages - Google Patents

Procede de selection et d'identification de molecules peptidiques ou proteiques par presentation a la surface de phages

Info

Publication number
EP1453959A2
EP1453959A2 EP02792779A EP02792779A EP1453959A2 EP 1453959 A2 EP1453959 A2 EP 1453959A2 EP 02792779 A EP02792779 A EP 02792779A EP 02792779 A EP02792779 A EP 02792779A EP 1453959 A2 EP1453959 A2 EP 1453959A2
Authority
EP
European Patent Office
Prior art keywords
viruses
ligands
molecules
spr sensor
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02792779A
Other languages
German (de)
English (en)
Inventor
Oliver Hill
Holger Ottleben
Stefan Dickopf
Klaus Burkert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Graffinity Pharmaceuticals AG
Original Assignee
Graffinity Pharmaceuticals AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10220602A external-priority patent/DE10220602A1/de
Application filed by Graffinity Pharmaceuticals AG filed Critical Graffinity Pharmaceuticals AG
Publication of EP1453959A2 publication Critical patent/EP1453959A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

  • the present invention relates to a method for the selection and identification of peptide or protein molecules by means of phage display.
  • Phage display screening systems follow this approach.
  • the combination used in wYro gene expression techniques with traditional biochemical approaches such as Affinity chromatography offers the possibility of functional gene selection by establishing a direct link between natural product affinity and the structure of the gene.
  • genes coding for non-viral proteins or peptides are incorporated into the viral genome in such a way that fusion proteins are generated between the desired non-viral protein or peptide and a viral coat protein.
  • the fusion protein is presented on the surface of the virus.
  • a typical phage display library a large number of DNA fragments which code for non-viral proteins or peptides are inserted into the viral genome. This is how viral particles are generated that present a multitude of proteins or peptides on the surface.
  • This phage display library is then brought into contact with a sample immobilized on a support.
  • viruses that present fusion proteins that interact with the immobilized sample to form a bond are retained on the support, whereas viruses that present non-interacting fusion proteins are washed away.
  • the interacting viruses are eluted and amplified by infection of a host culture. Repeated rounds of amplification and selection may be required to obtain a relatively homogeneous virus population that binds to the immobilized sample with high affinity.
  • the inserted DNA sections are then sequenced from individual virus clones and the amino acid sequences of the interacting proteins or peptides are derived therefrom.
  • the immobilization of an interaction partner is necessary to enable the separation of the virus-ligand complexes from the non-interacting viruses. It also facilitates process management, e.g. performing washing steps and, in conjunction with a suitable detection method, can provide information about the presence and the strength of the interactions between the interaction partners.
  • Spherical beads are often used as carriers for virus selection. Beads can, for example, be stacked up to form affinity columns.
  • a major disadvantage when using these bead affinity columns is that no spatial assignment of the beads is possible. This represents a considerable problem in the automation and miniaturization of bead-based phage display processes.
  • Another disadvantage is the complex handling of the beads, e.g. when performing washing steps.
  • Another disadvantage is that spaces form between the spherical beads, in which dead volumes of virus suspension accumulate.
  • a carrier for virus selection which ensures the immobilization of a large number of ligands and a universal and simple handling and thus enables automation and miniaturization of the method.
  • the geometry of the carrier used for virus selection plays an important role in the automation and miniaturization of the method. It is advantageous for this if the interaction partners immobilized on the carrier are arranged in a regular grid and positionally addressable. For the automation and miniaturization of the method, it would also be very advantageous if both the selection and the detection of binding events could be carried out on the identical surface. To enable the simple use of powerful robots for pipetting or the like, should the containers are also open at the top. A microtiter plate or a planar support (eg a membrane), for example, meet these requirements.
  • WO01 / 02554 describes a parallelized method for identifying interaction partners by means of a phage display, in which magnetic particles are used for the immobilization of the ligands. All selections are made in spatially separated volumes simultaneously in microtiter plates. The necessary transfer of the magnetic particles takes place automatically using a special magnetic array. The magnetic particles with the ligand-virus complexes are bound to the magnets of the array and transferred between the vessels. The discrimination between interacting and non-interacting viruses is carried out with an ELISA (Enzyme Linked Immunosorbent Assay) based procedure in a process following the selection.
  • ELISA Enzyme Linked Immunosorbent Assay
  • a major disadvantage of using microtiter plates as an incubation vessel is that the cavities only have a relatively small sample volume (e.g. 0.2 - 1.2 ml volume per cavity with commercially available 96-well microtiter plates; 20-60 ⁇ l with 384-well microtiter plates) allow for selection. Due to this volume limitation of the cavities, it is often necessary to enrich the viruses before screening. For example, phage titers between 1x 10 10 - 1x 10 11 pfu / ml (plaque forming units) are usually achieved with lysates of bacteriophage T7.
  • the screening process is thus negatively influenced by the solubility behavior of the viruses in the precipitate. Furthermore, the work steps associated with the precipitation of the viruses are time-consuming and difficult to automate.
  • a method is more advantageous in which the culture supernatants / lysates resulting from the multiplication of the phage library can be used directly in the screening process. This is made possible by using incubation vessels with a larger sample volume than the cavities of microtiter plates allow.
  • Another disadvantage is that a competing, simultaneous selection of structurally similar ligands against the phage library is excluded due to the separate volumes, since only one ligand can be immobilized per cavity. It is advantageous to select the interaction partners in a common sample volume, in which the immobilized interaction partners are arranged in a position-addressable, two-dimensional array. Suitable for this are planar supports (e.g. membranes), which are incubated in suitable large-volume vessels (dishes or tubes) or are themselves a vessel.
  • planar supports e.g. membranes
  • Hawlish et al. (Analytical Biochemistry 293, 142-145 (2001) describe a method for the selection of epitope-specific scFv fragments using an M13-based virus system.
  • a peptide array synthesized on cellulose membranes was used, which contains part of the primary sequence of the human C3a receptor in Form of fifty in the sequence overlapping, 15mer peptides represented. All viruses interacting with the array were eluted and propagated together after each selection round.
  • the identification of interacting viruses was carried out in a separate binding assay (ELISA) with the complete protein domain as ligand Some of the selected viruses were clonally separated after the fourth round of selection, 92 clones were propagated separately and analyzed in the binding assay, the increase in ELISA-positive compared to ELISA-negative viruses was assessed as a selection success, and an assignment of ELISA-positive viruses as binding partner to the individual Affinity ligands of the array used for the selection were carried out by further ELISA-based assays. For this purpose, the entire array was brought into contact with a single virus clone and the position of the Binding signal assigned the viral clone to one or more affinity ligands of the array.
  • ELISA binding assay
  • a separate assay must therefore be carried out against all affinity ligands contained in the array.
  • the viral clones were assigned to the individual affinity ligands immobilized in the array in a further binding assay (ELISA) which was carried out on the array.
  • ELISA binding assay
  • a disadvantage of this method is that a common ELISA for the identification of interacting viral clones is only possible when using peptidic affinity ligands which are derived from a common protein / polypeptide sequence.
  • a binding assay must be performed for each affinity ligand used in the array.
  • a disadvantage of using membranes as the surface is that the local concentration of the ligand can only be controlled with great effort. This can lead to the formation of unspecific virus-ligand complexes due to local avidity effects.
  • a very great disadvantage of the above-mentioned method is that the spatial information of the array with respect to the ligands is lost during the selection, since the interacting viruses i) cannot be detected on the array and ii) cannot be eluted from the array in a location-specific manner.
  • a measuring system is required with which the binding of the viruses during the
  • Selection can be detected and ii) a method is required by means of which bound viruses can be eluted from the array in a site-specific manner.
  • the selection and detection of the selection success must be able to be carried out in the same measuring system.
  • marker-based detection methods eg ELISA assays
  • the binding assay on which the selection is based is limited in its execution to the conditions of the labeling reaction, so that one is used for the selection advantageous variation of the binding assay, e.g. B. Changing the pH, the ionic strength or the use of detergents is not possible.
  • marker-based detection method used in the above-mentioned method is that the viruses identified in the selection process cannot be used for the further method steps.
  • marker molecules e.g. antibodies, streptavidin
  • these require a physical interaction between the ligand-virus complexes and the labeling reagent. This physical interaction can lead to a change in the binding between the ligand and the virus (weakening or strengthening) or to an impairment of the host-virus interaction (loss of infectivity).
  • marker-based detection methods it is to be expected that insoluble aggregates will form, so that the viruses contained therein are not available for further process steps.
  • a label-free detection method such as surface plasmon resonance (SPR)
  • SPR surface plasmon resonance
  • Biosensor immobilized ligands are described.
  • the proteins used as ligands lysozyme,
  • HM90-5, pB-1 each covalently coupled to the dextran matrix of a sensor chip and a limited volume of a phage library passed in a continuous flow over the sensor surface.
  • the viruses were subsequently identified with a
  • the viruses contained in the eluate were isolated and increased.
  • the primary selection success was the increase in the ratio of bindings to non-binders of the virus clones contained in the eluate, determined by ELISA.
  • the antibodies encoded by the interacting viruses were produced recombinantly and their dissociation constant compared to the ligand immobilized on the sensor chip was determined.
  • a very big disadvantage of this method is that a flow system is used. As a result, the arrangement of the sensor surfaces in a one-dimensional direction is predetermined (one-dimensional array).
  • the disadvantage here is that a two-dimensional sample arrangement (two-dimensional array) and its miniaturization are made significantly more difficult by the use of a flow system.
  • the invention is therefore based on the object of providing a highly parallelizable method for the selection and identification of peptide or protein molecules which can specifically interact with certain other molecules to form a bond, the disadvantages of the prior art being avoided or reduced.
  • the object of the invention is achieved by a method for the selection and identification of at least one representative (interaction partner) from a large number of peptide or protein molecules, which can specifically interact with at least one representative from a large number of molecules to form a bond, comprising the steps:
  • Virus presented interaction partners with the help of a label-free detection method with the help of a label-free detection method.
  • the ligands are immobilized in a two-dimensional array on a specifically designed solid phase support, which enables interaction partners to be detected without labels.
  • the selection and detection of the selection success can be carried out in one measuring system.
  • the detected interacting viruses can be treated further in subsequent process steps and, if necessary, increased, using either all bound viruses or only those bound to surface fields selected for the respective selection.
  • Another advantage of a label-free detection method is that the direct binding between the ligand and the virus is presented
  • Peptide or protein is detected. This is not the case when using marker-based detection methods.
  • the method according to the invention in conjunction with a suitable measuring system also enables parallel detection with a high integration density. This provides a highly miniaturized and parallelized phage display method, so that the detection for several or all ligands can take place in parallel.
  • Steps (a) and (c) are preferably carried out on the same surface of the solid phase support, the ligands being immobilized in a Cartesian grid (array) on the surface of the solid phase support, so that the position of each ligand is determined by its x and y coordinates is determinable on the array.
  • a large number of position-addressable surface fields also referred to according to the invention as ligand fields, can also be provided on the solid phase support, whereupon the ligands are immobilized.
  • the viruses not bound in step (a) are preferably removed in step (b) by elution.
  • the solid phase support contains a polymer-free surface on which the ligands are immobilized. Due to the very high protein adsorption resistance of this polymer-free surface, it is possible to have relatively weak interactions, i.e. To observe the binding between the ligand and the protein or peptide molecule presented by the virus, which in particular enables the use of low molecular weight ligands.
  • the label-free detection method is preferably based on an optical, electrical or vibration-based method.
  • An optical reflection method in particular surface plasmon resonance (SPR)
  • SPR surface plasmon resonance
  • the solid phase support can be designed as an SPR sensor surface support.
  • the host cells are preferably infected by the viruses bound to the surface of the solid phase support. This has the advantage that the binding between ligand and virus-presented peptide or protein does not have to be broken.
  • the method according to the invention can preferably be carried out in a device according to FIGS. 1 to 3.
  • FIG. 2 sectional perspective view and enlarged portion of a
  • FIG. 3 sectional view and a perspective view of a measuring area and associated sealing element.
  • the method according to the invention allows the selection and identification of one or more representatives of peptide or protein molecules from a large number of such molecules.
  • “representative” means that each different peptide or protein molecule does not usually occur as a single molecule in the multitude of molecules, but is present in the protein mixture in a more or less large amount.
  • the principle of selection and identification is then based on the fact that the peptide or protein molecule sought can interact with one or more previously selected “selection molecules” while forming a bond.
  • selection molecules are not particularly restricted in terms of their nature and can be of any structure, as long as they can be handled in such a test and are capable of forming a bond. According to the invention, they are therefore simply referred to as "molecules".
  • ligand is also used in the context of the present invention.
  • a peptide or protein molecule that is used for interaction, i.e. Binding to the ligand and capable of being selected and identified in this way is also referred to as an "interaction partner”.
  • the terms “identification” and “selection” mean an enrichment, preferably an individualization, of “interaction partners”. Consequently, both the identification of interaction partners in a large number or population of any number of different interaction partners, as well as the selection of individual partners in a previously enriched population are included.
  • the interaction between the interaction partner and the ligand which manifests itself in the form of a bond between the partners, can be characterized, for example, by a “key-lock principle”.
  • the interaction partner (peptide or protein) and the selection molecule (ligand) have structures or motifs that fit each other specifically, such as an antigenic determinant (Epitope) that interacts with the antigen binding site of an antibody.
  • the interaction partners on the surface of viruses are presented as peptides or proteins.
  • all peptides or proteins are included, the coding nucleotide sequences of which can be inserted into a viral genome. It is preferred that the expression of these peptides or proteins as part of the virus envelope allows an assembly of this envelope and thus a propagation of the virus.
  • the propagated virus is preferably infectious.
  • the interaction partners can also be presented on the surface of cells, in particular also bacterial cells or spores, as peptides or proteins.
  • peptides or proteins includes both natural and synthetic peptides or proteins.
  • natural proteins include antibodies, antibody fragments, receptors that interact with their specific ligands, peptide ligands that interact with their specific receptors or peptide domains that interact with specific substrates, including proteins and coenzymes, and other peptides or enzymes, etc.
  • forms of the aforementioned proteins or peptides produced recombinantly include, among other things, fragments of the proteins described above, which interact with specific ligands.
  • Synthetic proteins or peptides include both pseudogenes or fragments thereof expressed as well as proteins or peptides with a random amino acid sequence.
  • the peptides and proteins are thus preferably part of a library consisting of viruses, the viruses, preferably integrated into their genome, containing a nucleic acid sequence which codes for the corresponding peptide or protein.
  • This nucleic acid sequence is typically present in such a way that, when expressed, it leads to the synthesis of the peptide or protein as part of a fusion protein which consists of a coat protein of the virus or a part thereof and of the peptide or protein.
  • This fusion protein then has the ability to be localized on the surface of the virus and consequently to present the peptide or protein.
  • the term “ligand” describes molecules or compounds that are immobilized on the surface of a solid phase support.
  • the term includes macromolecules as well as “small organic molecules” Structural elements that can interact with peptides or proteins presented on viruses due to their structural peculiarities, and knowledge of the structure of the ligands can be used to draw conclusions about the possible structure or specific structural elements of the molecule presented on the virus.
  • macromolecules is understood to mean molecules with a high molecular complexity or a high molecular weight. These are preferably biomolecules, e.g. Biopolymers, especially proteins, oligo- or polypeptides, but also DNA, RNA, oligo- or polynucleotides, isoprenoids, lipids, carbohydrates (glycosides), and their modifications, as well as synthetic molecules.
  • biomolecules e.g. Biopolymers, especially proteins, oligo- or polypeptides, but also DNA, RNA, oligo- or polynucleotides, isoprenoids, lipids, carbohydrates (glycosides), and their modifications, as well as synthetic molecules.
  • receptors in particular come into consideration, but also proteins or peptides which represent epitopes or antigenic determinants of proteins.
  • the proteins can also be fusion proteins.
  • small molecules or “low-molecular molecules” is understood to mean molecules which are of less molecular complexity than the macromolecules defined above.
  • the term “small molecules” or “low molecular weight molecules” is not used uniformly.
  • WO 89/03041 and WO 89/03042 describe molecules with molecular weights of up to 7000 g / mol as small molecules. Usually, however, molecular weights between 50 and 3000 g / mol are given, but more often between 75 and 2000 g / mol and mostly in the range between 100 and 1000 g / mol.
  • small organic molecules is used for molecules with a molecular weight below 3000 g / mol, preferably below 1000 g / mol, most preferably below 750 g / mol.
  • oligomers or small organic molecules can be listed, such as oligopeptides, oligonucleotides, carbohydrates (glycosides), isoprenoids, lipid structures or haptens.
  • molecular weight is mostly the basis for the definition of such small organic molecules.
  • One aspect of the method or measuring system used according to the invention relates to the provision of a two-dimensional array with a large number of ligands on a solid phase support.
  • the ligands in the array are arranged such that the identity of each ligand can be determined by its x and y coordinates on the array.
  • the spatial structure of the resulting array can be predetermined by mechanical structuring of the carrier. If structured solid phase carriers are used in the present invention, they therefore preferably have a large number of regularly arranged, position-addressable fields (ligand fields). These ligand fields contain one or more cavities (sensor fields), on the bottom of which the ligands are immobilized. The cavities preferably have a depth of 20-100 ⁇ m.
  • ligand fields preferably differ in each case in the type of interaction partner immobilized on their sensor fields, it being possible for a single ligand field to present both a single ligand and several identical or different ligands.
  • four interaction partners are immobilized per ligand field.
  • the cavities are preferably arranged such that a regular, preferably Cartesian, grid of columns and strips is formed on the carrier.
  • the size and shape of the carrier can be chosen as desired and easily adapted to the detection system used.
  • the spacing of the fields from one another should preferably be adapted to the microtiter format used or the format of the spotting device used.
  • the number of fields on the solid phase support can also exceed the number of subunits of the microtiter plate, ie the density of the sensor fields on the solid phase support can be many times higher than the density of the subunits of the microtiter plate.
  • a rectangular solid phase support 6144 fields which can be occupied by a spotting robot from four conventional 1536 microtiter plates.
  • the method according to the invention is preferably carried out with an SPR sensor surface carrier as a solid phase carrier, which is divided into a multiplicity of measuring ranges, one measuring range each comprising one or more (e.g. four) SPR sensor surfaces. At least one of these measuring areas is surrounded by an insulating area, which does not include any separating means, and is designed to accommodate a sealing element in order to form, together with a volume element to be placed on the measuring area, a space isolated from adjacent measuring areas above this given measuring area.
  • This SPR sensor surface carrier makes it possible to create a volume over one or more measuring ranges, which is isolated from adjacent measuring regions, in order, for example, to carry out further measurements on samples directly on the SPR sensor surface carrier, which are located on the respective SPR sensor surfaces.
  • FIG. 1 a shows a first embodiment of the SPR sensor surface carrier, in which a multiplicity of measurement areas 110, which in the example shown each comprise four SPR sensor surfaces 100, are arranged on a prism 4, which in this example serves as the substrate of the SPR Sensor surface carrier is used. Also shown is a cuvette border 9, which is preferably attached around the overall arrangement of measuring areas 110. Also shown is a light beam 6 which passes through the prism 4 (the SPR Sensor surface carrier) is guided to excite a surface plasmon resonance in the SPR sensor surfaces 100.
  • FIG. 1 b shows a further embodiment of the SPR sensor surface carrier, in which a plate-shaped substrate 5 carries the SPR sensor surfaces 100 and the measuring areas 110. Similar to the embodiment in FIG. 1a, a cell border 9 is also provided.
  • this SPR sensor surface carrier it is applied to a surface 7 of a prism 4, so that light (more generally: SPR-exciting radiation) 6 can be guided through the prism 4 and the plate 5 to the SPR sensor surfaces 100 , This is preferably done by using an index liquid 8 between the prism 4 and the plate 5, so that the light 6 irradiated under SPR conditions is not reflected at the interface of the surface 7 at the air gap in front of the plate 5.
  • an index liquid is oleic acid or a mixture containing oleic acid.
  • any material that is transparent to SPR-capable radiation and onto which a SPR-capable material can be applied in the SPR sensor surfaces 100 can be used as the material for the prism 4 or the plate 5.
  • the substrate 4 or 5 can be made of glass, and the SPR sensor surfaces can be formed by a metal coating, in particular by a gold layer.
  • the measuring areas 110 can be addressed in two dimensions.
  • the term “addressable” means that individual measuring ranges can be distinguished from one another by means of a corresponding identification or address, so that corresponding samples can also be addressed accordingly. This creates the advantage that a very large number of measuring areas 110 can be exposed and evaluated simultaneously.
  • the measurement areas 110 are arranged in a Cartesian grid, as shown in FIG. 1, the addressability then is easiest given by Cartesian coordinates.
  • the present invention is in no way limited to this, and the measuring areas can be distributed in any grid or even completely disordered, and can be addressed regardless of their specific arrangement according to any coordinates (for example polar coordinates).
  • the carrier 5 is shown schematically, on which a gold layer 51 is located.
  • the measuring area 110 comprises four SPR sensor areas 100.
  • a measuring area can also include more or fewer SPR sensor areas 100.
  • the measuring area 110 is formed by suitable separating means 105 (examples of which are described later) which, as shown in FIG.
  • SPR sensor surfaces 100 are formed in which the gold layer is applied to the carrier 5 (possibly with an intermediate layer for promoting adhesion between the gold layer 51 and the carrier 5) and release agent regions in which the release agent 105 on the carrier or substrate 5 (also possibly with an adhesion-promoting intermediate layer ) are applied.
  • the separating means are designed in such a way that no surface resonance occurs there , so that the SPR sensor surfaces are clearly separated from one another by the structuring of the surface of the carrier 5.
  • the measuring region 110 shown is preferably surrounded by an insulating region 120.
  • the insulating area 120 is designed to accommodate a sealing element 130 in order to form an insulated space over this measuring area together with a volume element 11 to be placed on the measuring area 110 shown (see FIG. 3 above). It can be seen that the insulating region 120 does not comprise any separating means 105. This ensures that the sealing element 130 can provide a good seal.
  • the insulating region 120 on the surface facing away from the substrate 5 is preferably of the same nature as the SPR sensor surfaces. This can be seen in FIG. 3 above, since both the SPR sensor area and the insulating area 120 present the gold area 51. According to a preferred embodiment, not only are the surfaces made the same, but the SPR sensor areas and insulation areas are made the same overall, i.e. have the same layer sequence from the substrate 5 to the surface. In other words, the SPR sensor surfaces 100 and the insulating regions 120 are preferably produced by the same method steps, so that no separate method steps are necessary for the respective production.
  • the insulating region 120 on the surface facing away from the substrate 5 is of a different nature than the SPR sensor surfaces 100.
  • an insulating region 120 can be formed by an exposed substrate surface.
  • an insulating region 120 it is possible for an insulating region 120 to have a seal-promoting layer on its surface, which consists of a material that is matched to the sealing elements 130, e.g. Silicone. This seal-promoting layer can be applied in any desired or practical way, e.g. by means of a mask, by means of a robot that controls the individual insulation areas, or by hand.
  • the separating means 105 are raised not only with respect to the SPR sensor areas in order to form respective cavities on the SPR sensor areas, but also with respect to the insulating area 120, as shown in FIG. 3. With suitable dimensioning of the insulating region 120 and the sealing element 130, it is thus possible for the separating means 105, which form the circumference of the measuring region 110, to serve as a guide for the sealing element 130. The placement of the sealing elements 130 is thus facilitated.
  • the measuring areas 110 and the associated sealing elements 130 are preferably round or oval. However, it should be noted that the invention applies to any geometric shape of the The outer circumference of measuring areas and sealing elements can be applied.
  • FIG. 3 shows a single measuring area 110 with an associated insulating area 120.
  • FIG. 1 shows a plurality of measurement areas (as shown in FIG. 1) and only one or a few of these measurement areas are surrounded by associated isolation areas, it is preferred that each of the plurality of measurement areas 110 of a given SPR- Sensor surface carrier is surrounded by an associated insulating area 120. This has the advantage that a volume can be formed over any measurement area 110 by placing a corresponding sealing element and volume element in order to carry out further measurements and analyzes.
  • a method for producing an SPR sensor surface carrier according to the above-described embodiments is now set out. This is preferably done by forming or applying the release agent 105 on the respective substrate, e.g. of the plate 5 or the prism 4, so that free areas are created between the separating means 105, which define SPR sensor areas 110 and insulating areas 120, and then application of an SPR-suitable material at least in the free areas, which define SPR sensor areas 100.
  • an SPR sensor area carrier is created in which the insulating areas are characterized by the exposed substrate or the layer directly below the gold layer. If the SPR-suitable material is also applied in the free areas which define insulating areas, an SPR sensor surface carrier is produced, as is shown in FIG. 3, namely in which the SPR-suitable layer both in the SPR sensor surfaces 100 and in the isolation areas 120 is presented.
  • SPR-suitable material e.g. gold
  • a seal-promoting layer e.g. silicone
  • the step for forming the release agent 105 can be carried out, for example, by applying a polymer to the surface of the substrate 4 or 5.
  • This preferably comprises the steps of applying a photostructurable polymer to the entire surface of the substrate 4 or 5, exposing the applied polymer layer with a mask which defines regions which belong to the separating means 105, regions which belong to the SPR sensor surfaces 100 and areas associated with the isolation areas 120 and processing the exposed polymer layer to expose the substrate surface in the areas associated with the SPR sensor areas 100 and the isolation areas 120.
  • An alternative in applying a polymer for the release agent is to apply a polymer to the surface of the substrate 4 or 5 in a two-dimensional grid that defines the release agent 105, the SPR sensor surfaces 100 and the insulating regions 120, and the curing of the polymer.
  • the polymer is preferably applied using a screen printing technique.
  • the release agent can also be formed by a structurable silicon layer.
  • the step for applying the SPR-suitable material is preferably carried out by depositing a metal, an adhesion-promoting layer possibly being applied prior to the deposition of the metal. It is particularly preferred that the metal is evaporated on the entire surface of the structured substrate, so that it then also covers the release agents, as shown schematically in FIG. 3 above.
  • the present SPR sensor surface carrier is designed in such a way that the insulating area 120 can accommodate a sealing element 130 in order to form an insulated space above the measuring area together with a volume element 11.
  • the volume element 11 can be provided in any suitable or desired manner, for example as a cylindrical individual element which is connected to a single sealing element 130.
  • several volume elements 11 are provided as part of a volume element carrier 10, as shown for example in FIG. 2.
  • FIG. 2 shows a measuring device which consists of an SPR sensor surface carrier 5 and a volume element carrier 10 interact with each other so that 110 rooms are formed over the respective measuring areas.
  • the volume element carrier 10 is a body in which the volume elements 11 are formed as bores or recesses.
  • the volume element carrier 10 can e.g. by machining (e.g. milling or drilling), from a plastic (e.g. Teflon) or metal (e.g. aluminum).
  • Thermoplastics e.g. polystyrene or polypropylene
  • the volume element carrier can also be brought into the desired shape using a casting process (e.g. injection molding).
  • any suitable, moldable or solidifying material is suitable, e.g. the above-mentioned thermoplastic elastomers, such as polystyrene or polypropylene, or castable metals.
  • volume element carrier is to be used repeatedly in processes in which viruses, bacteria or other potentially infectious biological entities are used, preference is given to materials which are resistant to chemical sterilization (e.g. treatment with citric acid, NaOH / SDS).
  • materials which are resistant to chemical sterilization e.g. treatment with citric acid, NaOH / SDS.
  • Such a material is, for example, PolyChloroTriFluoroEthylen (PCTFE).
  • the sealing elements 130 are components of the volume element carrier 10.
  • the sealing elements 130 can be connected to the volume element carrier 10 in a fixed or detachable manner.
  • Grooves in which the sealing elements 130 are placed are preferably provided on the side of the body forming the volume element carrier around the openings which define volume elements 11.
  • the sealing elements are preferably O-rings.
  • the sealing elements and the volume element carrier can be formed in one piece. This is possible, for example, if the volume element carrier is injection molded from a suitable plastic is produced, which has sufficient flexibility for the sealing elements.
  • the sealing elements can be formed as protruding beads in a shape matched to the measuring areas (for example as ring beads for round or oval measuring areas) on the side of the volume element carrier which is to be placed on the SPR sensor surface carrier.
  • soft material seals are suitable as sealing elements, e.g. made of plastic, rubber, silicone, Teflon, etc., which can be used in ring, lamella or mat design. Vacuum seals can also be used.
  • the SPR sensor surface support and the volume element support 10 have respective adjustment elements, e.g. Dowel pins and guides 13 (see FIG. 2) so that the sealing elements 130 can be aligned with the assigned insulating areas 120.
  • the tolerances for the dowel pins and guides are matched to the dimensions of the measuring areas or insulating areas and sealing elements, so that the desired accuracy of fit between the sealing elements and insulating areas can be achieved.
  • the accuracy of fit of the dowel pins and guides is preferably on the order of 20 ⁇ m or less.
  • the SPR sensor surface carrier and the volume element carrier have respective fastening elements 15 in order to firmly connect the SPR sensor surface carrier and the volume element carrier to one another.
  • the fastening elements 15 are preferably such that the connection can also be released again.
  • the connecting elements 15 can e.g. be a pressure connection, such as a screw or clamp connection.
  • the fasteners can e.g. Be guides in which a metal clip is inserted to connect the SPR sensor surface support and the volume element support together.
  • the connecting elements can be internally threaded bores into which an outer screw is screwed in order to connect the SPR sensor surface support and the volume element support to one another.
  • the adjusting elements 13 and the fastening elements 15 are identical, which would be possible, for example, in the example of the threaded bores given above, since on the one hand the screwing in of the outer screw SPR sensor surface carrier and the volume element carrier connects, and on the other hand brings about an adjustment by aligning the holes.
  • the tolerances must create the required accuracy of fit. It is therefore preferred that the adjusting elements 13 and the fastening elements 15 are separate, since then the requirements for the tolerances in the fastening elements can be lower.
  • ligands In order to be able to increase the throughput during screening, it is advantageous to immobilize the largest possible number of ligands.
  • a number of at least 10, preferably 96, particularly preferably 384, very particularly preferably 1536, still more preferably 4608, most preferably 9216 different representatives of ligands can be immobilized.
  • immobilize the same ligand several times This can be useful, for example, for multiple determinations of the ligand-virus interaction in order to assess the quality of the selection process.
  • the figures given therefore only take into account the ligands which differ from one another. It must also be taken into account that different sites of the ligand can be used to immobilize the ligands.
  • the ligand thus has a different orientation on the surface and can sometimes show a different binding behavior. According to the invention, different orientations of a ligand are also understood as “different” ligands for simplification.
  • SPR surface plasmon resonance
  • the solid phase support can consist, for example, of a glass, plastic, metal, preferably a noble metal, particularly preferably gold, or has a layer of such a metal on the surface. This layer of metal can optionally be applied with the help of an intermediate layer, which serves to promote adhesion.
  • the material used to which the surface is applied depends on the measuring method used.
  • the solid phase support is preferably suitable for the use of label-free detection methods.
  • the solid phase support comprises at least two parts (see above), where one part can be made of at least partially transparent material and the other can be a metal layer.
  • it can be a glass body and a gold layer.
  • the carrier is preferably suitable for surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • a suitable sensor and measuring system is disclosed, for example, in WO01 / 63256, the full disclosure of which is hereby incorporated by reference.
  • the ligand can be immobilized directly or indirectly on the solid phase support. There are several ways of attaching ligands to a solid surface. A covalent, ionic or adsorptive bond can be mentioned here as an example. The covalent attachment of the ligand to the support is particularly preferred since this chemical bond is so stable that it permits complete denaturation of adhering proteins without impairing the surface properties.
  • the ligand can be used unchanged or chemically modified. Chemical modification involves changing existing reactivities, such as activating existing functional groups or adding another molecule that enables direct or indirect attachment to the surface. Simple addition or substitution reactions can be used for this.
  • An organic intermediate layer is advantageous in order to avoid or reduce the frequently occurring undesired unspecific binding of the ligand to the surface of the carrier, in particular if it consists of a plastic or metal surface.
  • a self-assembling monolayer (SAM) is often used here, which avoids adsorption of the ligand on the support. Self-assembly into a dense film is usually accomplished through the hydrophobic interaction of long chain hydrocarbons on them At one end there is a functional group that enables attachment to the support and the other end contains a functional group that enables the ligand to be attached. Connections that include these functional building blocks (head, foot group, hydrophobic part) are also called anchors. Furthermore, the anchor can have a spacer portion, which preferably contains ethylene glycol units, which ensure low non-specific protein adsorption.
  • diluent molecules are advantageously also added to the anchor molecules mentioned in order to control the concentration on the surface.
  • a too dense surface concentration can be disadvantageous due to steric hindrance.
  • Thinner molecules are structurally adapted to the anchor molecules, but they do not have a head group for the attachment of the ligand, since this should be avoided. Furthermore, they are usually shorter than the anchor molecules in order to avoid impairing the accessibility of the ligand for the peptide or protein presented on the virus.
  • a polymer such as e.g. Dextran
  • a polymer-free surface is preferred.
  • Another advantage of a polymer-free surface is that, due to the low non-specific protein binding, there is no need to use blocking reagents in the selection process. This is particularly advantageous since these blocking reagents also have non-specific protein binding, which is thus avoided.
  • Another advantage of polymer-free surfaces is that they can be regenerated very easily. For this purpose, reagents that enable the surface to be regenerated in one step (e.g. SDS-containing solutions or methanol-trifluoroacetic acid mixtures) can be used.
  • SAMs can be generated by chemisorption of alkylthiols on a metal surface (eg gold).
  • the long-chain molecules pack themselves onto the solid phase as SAM, with the gold atoms being complexed by the sulfur functions.
  • silanization of glass or silicon with reactive silanes containing epoxy or amino groups followed by acylation of the amino groups, for example with nucleoside derivatives (Maskos and Southern, Nucl. Acids Res. 20 (1992) 1679-84).
  • the application of the ligands to be immobilized is not limited to special processes. For more precise localization of the active areas on the surface, e.g. Conventional pipetting or spotting devices, but also stamping or ink jet processes can be applied.
  • non-interacting viruses in step (b) of the method according to the invention can be carried out using conventional methods known to the person skilled in the art.
  • the non-interacting viruses are preferably removed from the surface by elution.
  • the expression “non-interacting viruses” encompasses viruses which do not interact with the immobilized affinity ligand (s), ie do not bind to the ligand.
  • An elution process is, for example, a washing process.
  • the surface can be treated, for example, with suitable solutions, the composition of which ensures that the interaction of the interaction partner with the target molecule is not resolved.
  • step (c) of the invention The detection of the interaction between the ligands and the interaction partners presented by the viruses according to step (c) of the invention
  • the method can be carried out according to conventional detection methods known to the person skilled in the art, in which it is ensured that viruses which were detected in the selection process can be used in the further method steps. This is the case when using label-free detection methods.
  • the label-free detection of the interaction between the ligands and the interaction partners presented by the viruses in step (c) is based on an optical, vibration-based or electrical method.
  • the interaction is particularly preferably detected by reflection optics.
  • the interaction is preferably detected by determining the surface plasmon resonance (SPR).
  • a label-free detection method means that the same surface can be used both for the selection process and for the detection of binding events, which only requires a one-time surface evaluation and also enables an identical ligand presentation.
  • the sensor fields are imaged on a spatially resolving detector.
  • Each of the sensor fields can be used as a separate measurement surface, i.e. the binding of the phage particles can be detected separately for each sensor field.
  • the detector should be able to record all binding events in parallel and the detection itself should be done in parallel.
  • the detector is advantageously a CCD camera.
  • the light arriving at the intermediate regions of the ligand fields should be absorbed as much as possible, scattered away or directed away in a direction other than the detection direction. It is this contrast between the sensor field and the boundary that allows an assignment of the pixel areas in the image to a sensor field. About the pixels of an area in the image summed up during data acquisition, so that with good absorption of the intermediate areas, the spectra for the sensor fields also become more meaningful.
  • the detection step (c) can be followed by a further treatment step (d), which can comprise one of the following steps: (d1) elution of all bound viruses;
  • step (d1) the interacting viruses are eluted from the surface and to the
  • step (d3) host cells are added to the entire surface and infected by the interacting viruses, followed by elution of the infected host cells from the surface.
  • steps (d2) and (d4) only the viruses that have interacted with the ligands immobilized on certain selected sensor fields are treated as described above.
  • This is preferably achieved by a specifically designed lattice mask which is applied to the ligand field which contains the interacting ligand (s).
  • the cutouts of the grid mask are aligned in the same two-dimensional grid as the ligand fields on the carrier.
  • the adjustment of both grids is achieved by an adjustment device (eg dowel pins) in the grid mask and the carrier holder.
  • step (d2) an eluence is given into those recesses of the grid mask which enclose ligand fields which contain interaction partners interacting with viruses on their sensor fields, followed by the elution of the interacting viruses from the surface.
  • step (d4) host cells are placed in the recesses of the grid mask which contain sensor fields with which viruses have interacted, followed by the elution of the infected host cells from the surface.
  • step (d2) or (d4) the interacting viruses or infected host cells of several recesses are propagated together.
  • the method according to the invention further comprises a multiplication step (e) which is carried out after step (d):
  • the virus is propagated by diluting the infected cells in a pre-culture of the host strain and then growing the culture until lysis.
  • Conditions for propagating the interacting viruses by infection of a host are well known to those skilled in the art, for example ® in T7Select System Manual, Fa. Novagen, Madison (USA) (TB178 06/00), page 18 et seq.
  • step (b) the sequence of steps (a), (b) is repeated one or more times before the detection step (c) is carried out.
  • step (c) it is preferred to carry out the further treatment and multiplication steps one or more times, ie after step (b), the sequence of steps (d), (e), (a), (b) is repeated one or more times before the detection step (c) he follows.
  • step (c) can be carried out before step (d). The repetition of these sequence of steps ensures a selective enrichment of viruses that present interaction partners of the immobilized ligands on their surface.
  • the method further comprises a characterization step (f) which is carried out either after step (c) or after step (e):
  • any type of assay which is suitable for characterizing a binding can be considered as an assay.
  • Such an assay is preferably a solid phase assay.
  • Methods known in the literature are, for example, ELISA (enzyme-linked immunosorbent assays), RIA (radioimmunoassay) and surface plasmon resonance (SPR) or vibration resonance methods (Butler, J.E., METHODS 22, 4-23 (2000).
  • the binding is characterized in step (f) on the same or identical surface on which the virus population and the individual virus clones originating from this virus population have been identified and selected.
  • a preferred embodiment of the method further comprises the isolation and sequencing step (g):
  • Suitable methods are known to the person skilled in the art from the prior art which enable him to isolate and analyze the DNA sequences inserted into these, which code for the corresponding peptides or proteins, after the isolation of individual virus clones.
  • the method further comprises the recombinant expression and isolation or the chemical synthesis of the peptide or protein identified / selected as interaction partner of the ligand.
  • the method according to the invention further comprises characterizing the binding of the recombinantly expressed or chemically synthesized peptide or protein to individual ligands based on the selection of the ligands initially used in an assay.
  • the same assays are advantageously used as are used for checking the virus clones. This is useful in order to demonstrate that the selected interaction partner is not bound by the virus.
  • this characterization takes place on the same surface that was used for the identification / selection of the corresponding virus.
  • the interaction partners presented by the viruses are encoded by DNA fragments inserted into the virus genome, which form a DNA library.
  • the DNA library contains at least 10 2 , preferably 10 3 , even more preferably 10 4 , particularly preferably 10 5 , very particularly preferably 10 6 and most preferably 10 7 DNA fragments.
  • the inserted DNA fragments are isolated from cDNA or genomic DNA (gDNA) or are synthetic oligo- or polynucleotides.
  • the inserted cDNA or inserted gDNA preferably originates from a prokaryotic or eukaryotic organism.
  • the eukaryotic organism is preferably a fungus, a plant or an animal organism, preferably a mammal.
  • the mammal is preferably a mouse, a rat or a human.
  • the cDNA is isolated from a differentiated tissue or a differentiated cell population. Isolation of the cDNA from liver, brain, heart or breast tissue is preferred or cells. The tissues or cells preferably come from a healthy organism.
  • the tissues or cells come from a diseased organism.
  • the disease or suffering of the organism is preferably selected from the group consisting of cancer, hypertrophy and inflammation.
  • the viruses forming the virus system can include wild-type viruses and genetically modified viruses.
  • wild-type viruses and genetically modified viruses.
  • the virus system comprises a virus that uses eukaryotes as the host.
  • the virus system comprises a virus that uses prokaryotes as the host.
  • the virus can produce single-stranded DNA (ssDNA
  • Viruses or preferably selected from the group of viruses with double-stranded DNA (dsDNA viruses).
  • This dsDNA virus is more preferably selected from the group of bacteriophages.
  • Bacteriophages selected from the group of bacteriophages with tail, even more preferably selected from the group consisting of Myoviridae,
  • the bacteriophage is a for
  • the bacteriophage can also be a filamentous bacteriophage and is preferably selected from M13, f1 and fd phage.
  • a virus system comprising a lytic phage is also preferred.
  • This lytic phage preferably has a polyhedral, in particular an icosahedral, capsid.
  • the lytic phage is a ⁇ phage, a T3 phage, a T4 phage or a T7 phage.
  • the method according to the invention can be used, for example, for epitope mapping or for peptide lead structure identification. Furthermore, the method according to the invention is an ideal method to identify ligands that make purification steps more efficient.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
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  • General Physics & Mathematics (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de sélection et d'identification d'au moins un représentant (partenaire d'interaction) d'une pluralité de molécules peptidiques ou protéiques qui peut interagir spécifiquement avec au moins un représentant d'une pluralité de molécules cibles par formation d'une liaison, ledit procédé comprenant les étapes suivantes: (a) mise en contact d'un système viral composé d'une pluralité de virus parmi lesquels chaque virus présente à sa surface au moins un représentant de la pluralité de molécules peptidiques ou protéiques, avec la pluralité de molécules cibles (ligands) immobilisées à la surface d'un support en phase solide, adressables en position dans une trame tridimensionnelle; (b) élimination de la surface des virus qui ne se sont pas liés; et (c) identification des partenaires d'interaction par détection et détermination de la position de la liaison entre le ligand immobilisé et le partenaire d'interaction présenté par le virus, au moyen d'un procédé de détection sans marqueurs. Grâce à une répétition éventuellement cycliques de la sélection, le procédé de l'invention garantit une augmentation de la concentration en virus qui présentent des partenaires d'interaction. Eventuellement, des partenaires d'interaction sélectionnés sont exprimés de façon recombinée après identification de la séquence de nucléotides codante.
EP02792779A 2001-11-28 2002-11-20 Procede de selection et d'identification de molecules peptidiques ou proteiques par presentation a la surface de phages Withdrawn EP1453959A2 (fr)

Applications Claiming Priority (5)

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DE10158242 2001-11-28
DE10158242 2001-11-28
DE10220602A DE10220602A1 (de) 2001-11-28 2002-05-08 Verfahren zur Selektion und Identifikation von Peptid- oder Proteinmolekülen mittels Phage Display
DE10220602 2002-05-08
PCT/EP2002/013010 WO2003046198A2 (fr) 2001-11-28 2002-11-20 Procede de selection et d'identification de molecules peptidiques ou proteiques par presentation a la surface de phages

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US9562266B1 (en) 2012-01-14 2017-02-07 The University Of Toledo Amine-terminated aptamer functionalized surface plasmon resonanace sensors, methods of making and methods of using same
US20220018840A1 (en) * 2020-07-20 2022-01-20 Massoud Akhtari System and method for determining presence of certain attributes in a test article

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US20050014135A1 (en) 2005-01-20

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