METHOD FOR SCREENING LIGAND AFFINITY AND TOOL FOR USE IN THE METHOD

Abstract: Method for determining the affinity between a ligand, which is bound to a surface on a solid phase, and a target substance which is in a fluid, which method comprises at least two different adsorption experiments or at least two different desorption experiments which are similar with respect to one or both of the ligand and target substance and different with respect to at least one solid phase variable or at least one fluid variable. The method is characterised in that one uses a tool of the comb type with at least two essentially similar teeth on which there is a surface carrying a ligand.

Description: METHOD FOR SCREENING LIGAND AFFINITY AND TOOL FOR USE IN THE METHOD Technical field The technical fields of the invention are methods and tools for determining a) the solid-phase-linked affinity ligands for a predetermined target substance in a library of compounds (ligand candidates) and/or a target substance for a predetermined affinity ligand in a library of target substance candidates, b) suitable conditions (fluid variables) for affinity adsorption and/ or for desorption of an affinity adsorbing target substance, and c) suitable solid phase variables for affinity adsorption and desorption. Fluid variables comprise variations of, amongst others, the target substance. Solid phase variables include variations of, amongst others, ligands linked to the solid phase. The term "affinity binders" means that two substances (ligand respectively target substance) selectively/ specifically can bind to each other by affinity. The complex which is formed contains the ligand and the target substance. If the ligand is linked to a solid phase and the target substance is in solution then one speaks about affinity adsorption which implies that the complex is formed on the solid phase. The opposite reaction is called desorption and implies that one starts from a solid-phase-linked complex between the ligand and target substance and sets the conditions in the solutions so that the target substance is liberated from the complex and goes into solution. Examples of affinity ligands are groups which have a charge (anion exchanging, cation exchanging and amphoteric groups) amphiphilic groups, dipolar groups, bioaffϊne groups (antibody/ antigen/ hapten, Ig(Fc) -binding protein/Ig, lectin/ carbohydrate, complementary nucleic acids, etc), groups which are able to bind to each other via hydrophobic interaction, π-π interaction, tiophilic interaction etc. Affinity adsorption has been used in different chromatographic and batchwise processes. In order to optimize the ligand structure for a predetermined target substance one often performs several (> 1) more or less parallel experiments in which one varies the solid phase, ligand structure and chemistry in order to link the ligand to the solid phase etc. By varying the target substance and suitable target substances for a predetermined ligand structure. Through varying the conditions in the solution which contains the target substance one can find suitable conditions for adsorption. In a similar way in order to find suitable conditions for desorption one does the same but the starting 5 point is now the solid-phase-linked complex, which is brought into contact with different desorption solutions. In recent years experiments have involved one making more or less parallel test runs where the ligand and/ or target substance are allowed to vary, this 10 is popularly called the "screening" of libraries of ligands/ target substances. The individual ligands/ target substances are members of the library. In connection with the invention a library also means a number of different spacers, coupling groups, conditions for adsorption respectively desorption which are to be tested. Libraries have two or more members. 15 Earlier techniques where tools of the comb type were used By tools of the comb type is meant a tool which in a common holder has two or more projections (teeth) which in general have the same geometric shape 20 (in the main, essentially equally long, equally wide, etc]. The projections can in principle be arranged in different geometric patterns, for example one or more parallel rows, circles etc. Tools of the comb type have previously been used in order to perform, in parallel, one or more bioaffine reactions. The specific use has been in the context of quantitative or qualitative 25 determination of an analyte with the help of biospecific affinity reactions in assemblies of wells, for instance microtiter wells and wells in gels for electrophoresis. See US 5,618,671 ; US 5,618,701 ; US 5,759,784; GB 2, 118,738; GB 2, 147,698; WO 941 1529; WO 9418564; and WO 9635809. See also Fegler P., Zbl. Bakt. Hyg., I Abt. Orig. A 240 (1978) 1 12- 1 17 and 30 118-122. None of these publications concern screening for proper solid phase or liquid variables in a screening procedure as defined herein. The object of the invention 35 One of the main objects of the invention is to facilitate "screening" of the earlier mentioned types of libraries in order to find suitable combinations of ligand/target substance and other variables. In particular can be mentioned screening of a) libraries of ligands or target substances which are to be used in affinity adsorption in order to find suitable pairs of ligands /target substances, b) libraries of conditions in order to find suitable conditions in order to affinity adsorb a predetermined target substance to a predetermined ligand which is linked to a solid phase, and c) libraries of conditions in order to find suitable conditions for desorbing a target substance which in advance is affinity adsorbed to a solid phase which has a ligand structure which is capable of binding to the target substance. A partial object is to in a simple way be able to perform in parallel several test runs which differ among themselves with respect to at least one of the above-mentioned variables. Another partial goal is to increase the capacity of affinity adsorbents which are used during the "screening" of libraries in the technical field of the invention. These goals concern in the first instance to develop ligands and conditions for adsorption/desorption which can be used during purification methods which use chromatography matrices (see below). The invention A first aspect of the invention is a method as stated in the introductory part. This type of method involves making at least two different adsorption or desorption experiments which each comprise: i) that a) one contacts a ligand, which is stably linked to a surface on a solid phase with a fluid containing a possible target substance or b) one contacts a ligand which is stably linked to a surface on a solid phase and to which a target substance is preadsorbed, with a fluid the target substance can be released. ii) one investigates if adsorption respectively desorption of the target substance has occurred. During step i.a) above the target substance is dissolved in the fluid and is capable of binding to the target substance. The fluid used in step i.b) is preferably devoid of/ deficient in the target substance. The fluid is a liquid, typically water or an organic solvent including mixtures that if possible can be aqueous. That which is new in the method is that one uses a tool in the shape of a comb with at least two essentially equally long teeth which each have a surface carrying the ligand. If there are three or more teeth then these preferably are arranged in one or more rows (chiefly rectilinear) . In particular can be mentioned combs with 96 (like 12x8), 349 (like 16x24), or 1536 (like 22x48) teeth or other formats which are adapted so that the teeth fit in the individual wells in so-called micro titer plates. As special embodiments can be mentioned combs which have at least one row of teeth with 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 22, 24, 48 teeth per row. Substantially simultaneous contact between the fluid and surfaces is attained through the teeth of the comb being lowered into the fluid which is present in a well (container) which is common for all the teeth. In an alternative variant, the wells are arranged to permit at least two teeth to simultaneously be lowered into their own respective well, with the preference for that in the latter case there is one well arranged for each tooth. Variations (differences) between adsorption or desorption experiments can be achieved in two ways: a) through varying the solid phase or therewith linked substances (solid phase variables) and b) through different teeth being contacted with fluids which differ among themselves with respect to the amount and/ or type of constituent components (fluid variables). Examples of solid phase variables are the type of solid phase, coupling chemistry, spacers, ligand structure etc. Examples of fluid variables are pH, ion strength, type of ions present in the fluid, solvent, denaturing/ structure- decomposing agents, target substances, replacement agents, temperature, type of fluid (for example water, aqueous solutions, mixtures of solvents) etc. In the experiments one or both of the ligand and target substance are the same. In experiments which differ because of variations in one or more solid phase variables, the fluid which the surfaces are contacted with can be common for all the teeth or also a separate fluid can be used for each tooth. In the last case naturally it is also possible that one or more fluid variables vary between the experiments. In experiments which differ among themselves because of variations in one or more fluid variables separate fluids are used for respective tooth. This variation of the invention is principally intended for "screening" with respect to adsorption or desorption conditions or in order to find new target substances for one or more predetermined solid-phase-linked ligand structures. Naturally in this case variations in solid phase variables can also be used. The teeth surfaces which support the ligands according to the invention have preferably surface area enlargements in the shape of unevennesses or pores. "Surface area enlargement" means increasing of the surface area available for affinity adsorption. Suitable surface area enlargements are often increased by a factor of 2 or more, preferably more than ten, compared with corresponding plane surfaces without any unevennesses or pores. Surface area enlargement can be obtained through roughening of the surface, through attaching particles, which preferably are porous, to the surface, etc. For example, if the teeth have an outer plastic layer one can partially dissolve this with a solvent so that it becomes sticky whereafter one can contact the surface with particles, preferably in the form of a suspension. See further WO-A-9411421. In order that a pore system should serve its suitable purpose there should be individual pores which have such dimensions that the target substance can penetrate the system. This implies, as a rule, pore dimensions which are selected in the interval from 15 A and upwards. In order to speed up the adsorption process it can be good to have pore dimensions which permit convective mass transport. This implies, as a rule, pores which are greater than 1 μm. For practical reasons, as a rule, the pores are always less than 500 μm. For the case that the fluid which contains the target substance or which is to be used for desorption contains water then it is advantageous that the surfaces that shall come into contact with the fluid are hydrophilic. Thus the surface may carry hydroxyl, carboxyl, amino, polyethyleneoxyl, carboxamide or other polar or charged or chargeable groups. This applies to both exterior surfaces and surfaces in pores. For the case that the surfaces from the beginning are hydrophobic then they can be hydrophilized in a number of different ways. Known hydrophilization procedures are corona treatment, coating with a hydrophilic polymer, for example a polyhydroxyl polymer or another polymer which has a plurality of said hydrophilic groups, polymerisation to precipitate hydrophilic monomers etc. Examples of polyhydroxyl polymers which potentially can be used are polysaccharides, such as agarose, dextran, starch, cellulose, pullulan etc, and synthetic polyhydroxyl polymers, such as polyvinyl alcohol and polymers of hydroxyl alkyl (C2-10, chiefly C2-5) acrylate or methacrylate, hydroxyl alkyl (C2-10, chiefly C2-5) vinyl ethers etc. Said polymers can be cross-linked and/ or in some other way derivatised. In an advantageous embodiment of the invention the particles are built up from a polyhydroxyl polymer which preferably can be porous. As an alternative a porous hydrophilic surface has been formed through the surfaces of the teeth being coated with a polyhydroxyl polymer which gives suitable porosity. The ligand structure can be linked to the surface either covalently or via biospecific affinity, for example via biotin-strepavidin or via affinity between a high affine antibody and hap ten. Covalent bonding can occur via coupling with carboximide, halogencyan (for example BrCN), N-hydroxysuccinimidyl carboxylate, epoxide or halohydrine or vicinal dihalide, reactive squarate groups, thiol which possibly is activated etc. Between the ligand structure and the solid phase, an organic spacer can be positioned. The spacer can contain one or more straight, branched or cyclic saturated or unsaturated hydrocarbon chains (for example Ci-io, chiefly C1-5) which can be substituted with one or more hydrophilic groups according to the above. Ring structures can be aromatic even if this often is not preferred because of their hydrophobocity and possibility for undesired interactions. The individual hydrocarbon chains in a spacer may be linked together with each other via ether (-O-), thioether (-S-), disulphide (-S-S), ester (-CO-O-), amino (-NR-, where R can be hydrogen or lower alkyl (Cι-10, chiefly C1-5), aza (-N=N-), amide (-CO-NR-, where R can be hydrogen or lower alkyl (Ci-io, chiefly C1-5). Amongst further substituents on a hydrocarbon chain can be mentioned lower alkoxy, lower alkylthioxy (Cι-10, chiefly C1-5) and lower alkylamino (preferably C2-10, chiefly C2-5). The term spacer also includes the functional groups which link the spacer directly to the ligand structure respectively to the surface which is exposed to the fluid. Coupling of ligands to the solid phase often occurs in two or more stages. The first stage often involves one activating the solid phase so that reactive groups are introduced. Examples of popular reactive groups are activated hydroxyl or activated amino (for example activated with the help of halogencyan), thiol or activated thiol, N-hydroxysuccinimidyl carboxylate groups, activated squarate groups, epoxy or halohydrine or vicinal dihalide, etc. Another aspect of the invention is a tool of the comb type according to the above. The tool has a characteristic feature that on each tooth there is a surface, which has had its surface area increased by being made uneven and/or by having pores according to that which was mentioned earlier. A secondary aspect of the tool of the invention is that respective teeth surfaces are hydrophilised and possibly provided with reactive groups which can be used in order to link ligands. Among reactive groups can be mentioned specially those which were discussed in the foregoing paragraph. To determine if a target substance candidate has been affinity adsorbed takes place in a manner known in itself. Consequently after the adsorption stage and the removal of the fluid, which initially contains the target substance candidate, one can investigate the presence of the target substance directly on the surface. Alternatively one contacts the surface with a desorption solvent, whereby the subsequent occurrence of the target substance in a desorption solution is an indication that the target substance earlier was affinity adsorbed to the surface. The fluids' capacity (fluid variables) for the desorption of a target substance occurs through determining the presence of the target substance in the respective fluid after that it has been contacted with the surface which contains preadsorbed target substance. The chief advantages of the invention are obtained during the compilation of ligands and conditions for adsorption/ desorption, which are to be applied to purification methods which use chromatographic affinity matrices in packed and expanded beds and in batchwise methods. The advantages depend to a large extent on that the invention renders it possible in a simple way to imitate and vary the different conditions which are used in these types of purification methods (matrices, coupling chemistry for ligands including possible spacers, conditions for adsorption/ desorption, etc). The target substances and ligands can have one or more structural elements selected from for example polypeptide, nucleic acid, hydrocarbon and lipid structures. They can be of completely synthetic origin but can also be partly or completely made from naturally occurring compounds (biomolecules) . The invention shall now be illustrated with examples of practical embodiments which show how one "screens" in order to find suitable pairs of target substances and solid-phase-linked ligand structures, suitable adsorption conditions and suitable desorption conditions. EXPERIMENTAL PART Example 1: Screening of the binding capacity for different ligands for the one and the same target protein Coupling of ligands: A comb of polystyrene with a number of teeth coated, in accordance with WO-A-9411421, with N-hydroxysuccinimidyl activated HP-Sepharose (HiTrap® NHS-activated, Amersham Pharmacia Biotech AB, Uppsala, Sweden) (lμl gel/tooth) is coupled to different ligands which have NH2 groups. The gel contains agarose particles which are spherical, porous, hydrophilic and cross-linked (Sepharose® High Performance, dpso = 34 μm) and preactivated with N-hydroxy-succinimide for the coupling of proteins. The ligands were dissolved in DMSO. The coupling involved each tooth of the comb being dipped in a separate well containing 15 μl ligand solution (7 mg ligand/ml DMSO). After coupling (60 min) the uncoupled ligand was washed away with DMSO and thereafter the teeth were washed with distilled water. Remaining unreacted NHS-groups on the teeth were deactivated in buffer (0, 1 M Tris 0, 15 M NaCl pH 8). The teeth were then allowed to stand in a buffer for approximately 5 min before they were transferred to the actual adsorption buffer (20 mM PBS, 0, 15 M NaCl pH 7). The teeth were then dipped in wells containing the target protein (BSA) dissolved in the adsorption buffer (10 mg BSA/ml buffer) and were allowed to stand in the wells for approximately 30 min. Thereafter the unbound protein was washed away with adsorption buffer. Detecting with electrophoresis: The comb was placed with its teeth standing in a horizontally cast akrylamide gel with wells which precisely fitted the teeth. The gel contained a suitable desorption buffer for the target protein (0, 1 M Tris-glycine, pH 8,8, 0,2% Triton X100 10% isopropanol). After approximately 10 min the combs were removed and electrophoresis started. The run took approximately 1 hour. The gel was developed in an Autostainer with Commassie blue whereby the gel was coloured blue at the positions which corresponded to the teeth to which a BSA-binding ligand had coupled via reaction with the NHS-activated gel. Example 2: Screening for determining suitable desorption buffers Adsorption/ desorption: A comb to the teeth of which a ligand (according to example 1) which binds BSA had been coupled, was incubated in buffer (20 mM PBS, 0, 15 M NaCl pH 7) (15 min). Each tooth was then dipped in a radioactively marked protein solution (10 mg BSA/ml, 20 mM PBS, 0, 15 M NaCl, pH 7) whereby BSA was bound to the teeth of the comb. Through being dipped in wells the different teeth were contacted with different desorption buffers. The teeth were then washed with distilled water. Detection: Bound protein (BSA) remaining on the individual teeth can be determined through the teeth being cut from the comb and placed in "scint" cans containing the scintillation fluid. Thereafter the screening of radioactivity for each tooth can be determined in a scintillation counter wherein the number of counts is proportional to the quantity of BSA which remains on the teeth. The tooth which has the lowest value of protein content has been exposed to the most effective desorption buffer. Example 3. Screening for determining suitable adsorption buffers Adsorption: Ten combs each having 6 teeth (two of the teeth being coupled with a ligand which is known to bind BSA/HSA, two with a ligand of which the binding capacity to BSA/HSA is unknown and two which lack ligands (blanks)) were dipped in 10 different adsorption buffers each containing 10 mg HSA/ml buffer. The combs were incubated for 15 min before they were washed with the respective buffer. The coupling of the ligands occurred in accordance with example 1. Detecting with electrophoresis: The combs were placed standing in a horizontally moulded akrylamide gel with wells which fitted the teeth positions. The gel contained the suitable desorption buffer for BSA/HSA (0, 1M Tris-glycine pH 8,8, 0,2% Triton X100 10% isopropanol) . After approximately 10 min the combs were removed and electrophoresis started. The run took approximately 1 hour. The gel was developed in an Autostainer with Commassie blue. The gel was coloured blue in the positions that corresponded to teeth to which the HSA had bound which indicated that the adsorption buffer for these teeth functioned. Result: The known BSA/HSA-binder showed good affinity in all the buffers. The ligand with unknown binding capacity showed affinity in two of the buffers. The blanks showed no affinity in any of the buffers. Example 4: Screening of the binding capacity of different proteins for a certain ligand in the same buffer Adsorption: The respective tooth on a 7-tooth comb where each tooth is coupled (in accordance with example 1) with one and the same HSA/BSA- binder (ligand) were dipped in 7 separate wells containing 7 different proteins (HSA/BSA, ovalbumin, transferrin, β-lactoglobulin, serum and blanks) dissolved (10 mg/ml) in buffer (20 mM glycine pH 9). The teeth were incubated in the test solutions for approximately 15 min. Thereafter the teeth were washed with buffer (20 mM glycine pH 9) in order to remove non- bonded sample. Detecting with electrophoresis: The comb was placed standing in a horizontally cast akrylamide gel with wells which precisely fitted to the teeth. The gel contained the suitable desorption buffer for the protein (0, 1 M Tris-glycine pH 8,8, 0,2% Triton X100 10% isopropanol). After approximately 10 min the combs were removed and the electrophoresis started. The run took approximately 1 hour. The gel was developed in an Autostainer with Commassie blue. The gel was coloured blue in the positions which corresponded to teeth where the protein had bonded which indicated that the ligand had affinity to the protein in question. Result: In the buffer (20 mM glycine pH 9) HSA/BSA and proteins in the serum sample bonded to the tested ligands. The other samples showed no affinity.

Claims: CLAIMS Method for determining the affinity between a ligand, which is linked to a surface on a solid phase, and a target substance, which is in a fluid, which method comprises at least two adsorption experiments or at least two different desorption experiments which are similar with respect to either one or both of the ligand and target substance and different with respect to at least one solid phase variable or at least one fluid variable, which method is characterised in that one uses a tool of the comb type which has at least two essentially similar teeth on which there is surface carrying a ligand. Method according to claim 1 , characterised in that the experiments are different with respect to a solid phase variable selected amongst a) type of solid phase, b) coupling chemistry for linking the ligand structure to the solid phase, c) spacer, d) ligand structure, or a fluid variable selected amongst e) type of fluid, f) pH, g) temperature, h) ion strength, i) ion type, j) denaturing agent, k) replacement agent etc. Method according to any of the claims 1 - 2, characterised in that the respective surfaces, which support the ligand structures, are built up of a plastic support the surface of which is enlarged, for example through the surface being provided with unevennesses and/ or being porous. Method according to claim 3, characterised in that the surface enlargement is obtained through particles, which possibly preferably are porous and the outer and/or inner surfaces of which typically are hydrophilic, being bound to the surface. Method according to claim 3, characterised in that the surface area enlargement is obtained through the surface being porous with pore surfaces which typically are hydrophilic. Method according to any of the claims 3 - 5, characterised in that the respective surfaces have a polyhydroxy polymer to which the ligand is bonded. Method according to claim 6, characterised in that the polyhydroxy polymer is selected from amongst polysaccarides, such as agarose, dextran, starch, cellulose, pullulan etc which possibly can be cross- linked or in some other way derivatised, and synthetic polyhydroxy polymers, such as polyvinylalcohol and polymers of hydroxyalkyl (C2-10) acrylate or corresponding methacrylate, hydroxyalkyl vinyl ethers etc. Tool for simultaneously, at least qualitatively, determining the possibilities for affinity adsorption between one or several solid-phase- linked ligands and one or several target substances or for determining conditions for: a) affinity adsorption between a target substance and a ligand which is stably bound to a solid phase; or b) desorption of said target substance after that it has been affinity adsorbed to said ligand, characterised in that the tool consists of a comb with at least two teeth, which are arranged in rows and on each and every one of which there is a surface which has a stably bound ligand or a reactive structure to which a ligand can be linked covalently before the determination begins. Tool according to claim 8, characterised in that at least one solid phase variable selected amongst a) type of solid phase, b) coupling chemistry for binding ligands to the solid phase, c) spacers, d) ligands is different for at least two teeth. Tool according to any of the claims 8 - 10, characterised in that the surfaces are constructed in the way as defined in the method claims 3 - 7. Tool according to any of the claims 8 - 10, characterised in that it has at least one row with teeth and that there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 22, 24 or 48 teeth in each row.

Inventors: GLAD GUNNAR, NORRMAN NILS, GLAD, GUNNAR, NORRMAN, NILS
Applicants: AMERSHAM PHARM BIOTECH AB, GLAD GUNNAR, NORRMAN NILS, AMERSHAM PHARMACIA BIOTECH AB, GLAD, GUNNAR, NORRMAN, NILS
Application year: 1999
Main classification: G01N33
IPC codes: G01N30, G01N33