Method for the production of a solid phase matrix

0001The invention relates to a method for producing a specifically bindable substance bound to an insoluble carrier material, in particular for use in a heterogeneous analysis method based on the principle of the immunoassay.

0002To determine a specifically bindable substance, methods based on the principle of the immunoassay are often used. One of the partners of a substance pair that is specifically capable of binding to one another is reacted with the receptor specific for it, which is labeled in a manner known per se. The conjugate from these two substances can then also be reacted with a receptor which is specific for the conjugate or one of the two parts of the conjugate. There are many variations for these immunological procedures. It is advantageous if one of the receptors is bound to a solid phase. This facilitates the separation of bound and unbound reaction partners. To determine the specifically bindable substance, the amount of labeled reactant bound to the solid phase or of labeled reactant present in the solution is then measured and related to the amount of reactant to be determined in a manner known per se.

0003Plastic tubes or microtiter plates, on the inner surface of which the reaction partner is fixed, or spheres, on the outer surface of which the reaction partner is fixed, are usually used as the solid phase in the immunological processes. These plastic tubes, microtiter plates or spheres usually consist of relatively inert plastic material, so that the binding of the reactant is difficult.

0004In addition, the binding of the specific reaction partner to the respective surface must take place in such a way that it does not lose the ability to bind specifically the substance capable of binding specifically to it. For this reason, the binding of the reactant to the solid phase is mostly adsorptive. It has therefore already been proposed to fix the reactant to the solid phase using a coupling agent which mediates the binding. Care must again be taken to ensure that the binding of the reaction partner to the coupling agent does not destroy the specifically reacting region of the molecule or that the reaction partner is bound so that its reactive site faces the binding partner away from the solid phase. Furthermore, in DE-OS 25 33 701 it is proposed in order to achieve better binding, crosslink the individual immunologically active proteins and then adsorb them on polystyrene balls. A further possibility is given in this literature reference to crosslink an inert protein simultaneously with the protein with immunological properties, so that a crosslinked product is formed from inert and active protein, which in turn is then adsorbed on polystyrene beads. However, depending on the reaction conditions chosen, this type of crosslinking leads to different, non-reproducible crosslinkings with fluctuating proportions of non-crosslinked and insoluble protein. The different degrees of crosslinking also result in products with different binding properties. A similar process is described in EP-A-122 209 and also has the same disadvantages. All of these known processes are therefore unsatisfactory, do not yet lead to optimal adhesion of the specifically bindable substance and are not very suitable for the reproducible production of coated solid phases.

0005Another problem is also that the antibody bound to the solid phase usually has to be different for different tests. A specifically coated solid phase must therefore be made available for each test. This is very time-consuming. In addition, since there is no guarantee that any antibody immobilized in a known manner remains capable of binding, since unspecific bindings often also occur, and since the rate of detachment is also very high in the processes known hitherto, the antibody must be used in high excess. Nevertheless, the number of binding places is limited. This results in an increased susceptibility to faults. This is disadvantageous because antibodies are difficult and expensive to produce.

0006From WO-A-8 704 794 a solid phase matrix is ​​known which consists of an inert particulate polymeric carrier to which a crosslinked avidin layer is adsorbed via biotin. This arrangement cannot prevent bleeding.

0007From EP-A-0 245 926 a method for the determination of antigens, antibodies or haptens is known, in which a soluble complex of the sought substance with a partner capable of binding therewith and a soluble matrix is ​​formed in the liquid phase, and then the complex is made insoluble on a solid support by binding a ligand bound to this matrix to an anti-ligand and then the insolubilized complex is determined in the usual way by means of a label. This process is not secured against detachment of the soluble complex from the insoluble carrier.

0008It was therefore an object of the invention to provide a process which reproducibly improves the adhesion of the specifically bindable substance to the solid phase and, moreover, to provide a process with which a solid phase matrix can be produced which can be used universally for all immunoassays. Since many immunological processes are carried out with the addition of detergents in order to avoid clouding and non-specific binding, it was also the object of the invention to improve the adhesion to such an extent that the bound, specifically bindable substance does not become detached even in the presence of detergents.

0009This aim is achieved by a process for the production of a specifically bindable substance bound to an insoluble carrier material, in particular for use in a heterogeneous analysis process according to the principle of the immunoassay, which is characterized in that a first polymer I, which consists of a large number of partners P 1 of a specific binding pair and a water-soluble biological polymer or derivative thereof with a molecular weight of more than about 20,000, binds to an insoluble carrier material and with a second polymer II, which consists only of a large number of molecules of the other partners P₂ of the specific binding pair or from P₂ which are cross-linked with the other components, cross-linked via the specific binding of P 1 with P₂, the polymer II has both binding sites for P₁ and for an immunologically complex to be detected.

0010This method can be used to produce a solid phase matrix which can be used for all types of immunoassays, for example sandwich tests or competitive tests in a one-step or two-step process. For the sandwich test in a one-step process, for example, polymer II from partner P₂ can provide the binding site at which the complex to be determined is then immobilized. During the determination process, a sample which contains the substance to be determined is reacted with a labeled receptor and an unlabeled receptor which is capable of binding to polymer II. The complex formed from the substance to be determined, labeled receptor and receptor with a binding site for polymer II then binds to polymer II on account of the specific binding ability, so that the entire complex is immobilized in this way. After the phases have been separated, the marking can then be measured in one of the two phases.

0011If the sandwich test is carried out in a two-step process, a solid phase matrix can be used in which an unlabeled receptor is bound in the polymer II. The sample and the labeled receptor are then incubated in the presence of this solid phase matrix. After the phases have been separated, the label bound to the solid phase can then be measured.

0012When performing a competitive test, the sample and labeled sample analog compete for an unlabeled receptor. In this case, a polymer II to which the unlabeled receptor is bound is used for the test variant of the one-step process. For the other variant of the two-step process, the unlabeled receptor is bound in competition with the labeled receptor during the test, so that in this case a polymer II is used which has binding sites for these receptors.

0013To produce the solid phase matrix according to the invention, an insoluble carrier material is coated with a first polymer I, the binding to the carrier material not being covalent, but being brought about by adsorption or by interaction, and crosslinked with a second polymer II. Biological polymers which are water-soluble and have a molecular weight of more than about 20,000 are suitable for the polymer I. Proteins, peptides, nucleic acid polymers, carbohydrates and copolymers of amino acids and carbohydrates are preferably used for the polymer I. Derivatized polymers such as carbohydrates in derivatized form, for example aminodextran, are also suitable. The polymer I furthermore has a large number of partners P 1 of a specific binding pair. The partners P₁ can be cross-linked with the polymers or bound to them. The polymer I has a molecular weight of more than 20,000, since binding to the insoluble carrier material can no longer be guaranteed if the molecular weight is lower. The molecular weight is preferably more than 45,000 and particularly preferably more than 200,000.

0014The binding of the partner P₁ to the polymer takes place in a manner known per se. Suitable coupling methods are, for example, in Ishikawa, J. Immunoassay,<u style="single">4</u>, 209-327 (1983). Either functional groups of the partner P 1 which are suitable for binding to the polymer are used, or if suitable functional groups are not present, these are introduced into the P 1 molecule. For example, when using biotin as P₁, the N-hydroxysuccinimidyl derivative can be bound by reaction with amino groups present in the polymer. Other suitable derivatizations are known to the person skilled in the art and need not be explained here.

0015The ratio of the partner P 1 to the polymer used for the polymer I is not critical per se and can be varied within wide limits. It has been found that it is advantageous to use 1 to 200 mol of partner P 1 per polymer. The P₁ amount is dependent on the intended use and the polymer used. Since the polymer I as a whole should be relatively hydrophobic in order to be able to effect an adsorptive bond, if P₁ is hydrophilic, the proportion of P₁ should be lower, the smaller the molecular weight of the polymer used. This is the case with biotin, for example.

0016Suitable binding partners that can be used as P₁ and P₂ and in the binding between the polymer II and the immunologically detectable complex are, for example, biotin-avidin, biotin-streptavidin, antigen-antibody, hapten-antibody, protein A-immune-γ- Globulin and protein G-immune-γ-globulin. Antigen or hapten are also understood to mean conjugates of proteins with antigen or hapten or fragments thereof. The antigen can itself be an antibody, its Fab, Fab 'or (Fab') ₂ fragment. Antibodies are understood to mean monoclonal and polyclonal, complete antibodies and antibody fragments. Is used as P₁ or P₂ protein A or Protein G used, so in the immunoassay only the one receptor to be bound to the solid phase should be a complete antibody, while a Fab or F (ab ') ₂ fragment should be used as the labeled receptor, in order to avoid any non-specific Cause binding of the labeled receptor to the solid phase, which would lead to a falsification of the result.

0017A protein is preferably used for the preparation of the polymer I, which is more hydrophobic than the partners P₁ and P₂. Soluble proteins with a molecular weight of about 200,000 to about 20,000,000 are particularly suitable, which were optionally obtained from proteins with a molecular weight of 10,000 to 700,000 and are conjugated with partners P 1 of a specific binding pair.

0018Molecular weight and hydrophobicity are determined by the methods known to the person skilled in the art. The following methods are suitable for comparing the hydrophobicity between soluble protein and specifically bindable substance:<ul id="ul0001" list-style="dash"><li>fluorescence quenching after binding to dyes (Biochem. Biophys. Acta <u style="single">624</u>, (1980), 13-20),</li><li>the elution behavior in hydrophobic chromatography (Biochem. Biophys. Acta, <u style="single">576</u> (1979), 269-279),</li><li>the surface tension (Biochem. Biophys. Acta, <u style="single">670</u> (1981), 64-73),</li><li>retention times in Hydrophobic Interaction Chromatography (HIC) (Angew. Chemie <u style="single">98</u> (1986) 530-548, J. Chromat. <u style="single">296</u> (1984) 107-114, Anal. Biochem.<u style="single">137</u>, (1984) 464-472).</li></ul>

0019A comparison of the hydrophobicity of substances suitable according to the invention can be found in Sep. Sci. Technol.<u style="single">14</u>, 305-317 (1979). Then the hydrophobicity increases, for example in the following series: α-₂ macroglobulin (MW 820,000) Bovine Serum Albumin / Human Serum Albumin (MG 70,000) Egg albumin α₂HS glycoprotein (MW 49,000) β_1c/ β_1AGlobulin Immunoglobulin (MG 150,000) Transferrin (MG 90,000) Thus, if an immunoglobulin is used as the specifically bindable substance, human serum albumin or α₂HS glycoprotein, for example, are not suitable as soluble proteins without further pretreatment for this specific embodiment.

0020Both proteins have to be subjected to both hydrophobization and an increase in molecular weight. In this case, crosslinking is sufficient for transferrin, and hydrophobization is sufficient for α₂-macroglobulin.

0021Proteins which are suitable for coupling with immunoglobulin as a specifically bindable substance without pretreatment are, for example, β-lipoproteins (MW approx. 3.2 million) or α₂-lipoprotein (MG approx. 5 to 20 million).

0022The hydrophobization can be carried out, for example, by applying heat, treatment with acids, denaturing agents and / or chaotropic ions and / or by chemical coupling with a hydrophobic compound.

0023The molecular weight can be increased, for example, by applying heat, treatment with acids, denaturing agents and / or chaotropic ions and / or by crosslinking with a bi- or polyfunctional compound.

0024A protein which is not sufficiently hydrophobic or whose molecular weight is not sufficiently high is treated until a protein polymer having a molecular weight of 20,000, preferably 45,000 and particularly preferably more than 200,000 is obtained. A protein polymer with a molecular weight of 500,000 to 20 million is very particularly suitable.

0025If the protein is to be crosslinked, hydrophobization can take place before, during or after the crosslinking. However, the hydrophobization cannot be carried out in the presence of the specifically bindable substance if the specifically bindable substance is a protein and loses its biological activity as a result of the hydrophobization.

0026For hydrophobization by heating, temperatures of 40 to 95 ° C. are usually used in a period of 1 min to 10 hours, as in Biochem, for example. Biophys. Acta<u style="single">624</u> (1980) 13-20.

0027Acetic acid, propionic acid, lactic acid or hydrochloric acid, for example, are suitable for treatment with acids. Usual concentrations are 1 to 100 mmol / l with exposure times of 10 min to 16 hours.

0028For the treatment with chaotropic ions, for example, thiocyanates, iodides, fluorides, bromides, perchlorates and sulfates are suitable. For example, guanidine hydrochloride or urea can be used as denaturing agents. Usually concentrations from 10 mmol / l to 6 mol / l are used.

0029For the derivatization of the polymer I with hydrophobic compounds, soluble fatty acids, lipoids in low or high molecular weight form and synthetic polymers such as polypropylene glycol or soluble copolymers of polystyrene are preferably used. The derivatization takes place according to the methods familiar to the person skilled in the art.

0030The crosslinking is carried out with bifunctional or polyfunctional compounds. These are compounds which carry at least two functional groups, which can be the same or different, and which can react via these functional groups with functional groups of the compounds forming the polymer I, for example of proteins. Compounds are preferably used which consist of an alkyl chain, at the ends of which are succinimide, maleimide and / or aldehyde groups.

0031The crosslinking is then carried out with the bifunctional or polyfunctional compound in a manner known per se.

0032Proteins with a molecular weight of 10,000 to 700,000 are preferably used for the hydrophobization and / or crosslinking. Bovine serum albumin, lipase or immune-γ-globulin is particularly preferably used.

0033The polymer I prepared in this way is then bound to an insoluble support material. The binding takes place via the polymer and is usually adsorptive. The commonly used solid phases such as luran, glass, titanium dioxide, polystyrene, γ-activated polystyrene, polystyrene-acrylonitrile copolymer, paper and / or polyolefin are suitable as the carrier material. The carrier material can be physically or chemically pretreated before further processing. For example, a plastic surface can be pre-swollen or activated in another manner known per se. The carrier material is usually in the form of tubes, microtiter plates or spheres. However, other configurations are also possible.

0034The insoluble carrier material can also be an already coated material. For example, tubes pre-coated with polystreptavidin or tubes pre-coated with polymerized antigen are suitable. In this case, the insoluble carrier material provides binding sites for the polymer I, so that the binding of the polymer I then takes place not only by adsorption. A very strong bond is obtained in this way. In the case of the adsorptive coating, the polymer I is not very strongly bound. However, this is sufficient for most applications, since even when a few P 1 are detached, the wall adhesion is stabilized by the crosslinking with the polymer II. A stronger bond may be desired for special purposes. In this case, a pre-coated material can be used as the insoluble carrier material.

0035The polymer I is crosslinked with a second polymer II, which has a large number of partners P₂. The polymer II can either consist only of P₂ or a mixture of P₂ and other components. The polymer II has not only binding sites for P₁, which are supplied by P₂, but also binding sites for an immunologically detectable complex, which will be referred to below as a receptor. The receptors used are specifically bindable substances, in particular either specific bindable complete antibodies, which can be polyclonal or monoclonal, their antibody fragments or conjugates of antibodies or antibody fragments with haptens or antigens as well as haptens or antigens or binding proteins (such as thyroxine-binding globulin). The binding site for the receptor can either also be supplied by P₂ or by another component of the polymer II.

0036The individual partners P₂ can be connected to one another via homo- or heterobi-or -polyvalent compounds (linkers). Crosslinking with bivalent linkers is then preferably carried out, since this enables easier control of the degree of polymerization. However, polyvalent linkers are also suitable. Compounds which have reactive groups and which are able to react in aqueous solution with the functional groups of the specifically bindable partners to form a covalent bond can be used as linkers. A large number of suitable bifunctional or polyfunctional linkers are known to the person skilled in the art. Typical examples of homo- or heterobifunctional and trifunctional linkers which are very suitable in the context of the invention are listed in Table 1 below. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Short name</entry><entry namest="col2" nameend="col2" align="center">Chemical name</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">SPDP</entry><entry namest="col2" nameend="col2" align="left">N-succinimidyl 3- (2-pyridyldithio) propionate</entry></row><row><entry namest="col1" nameend="col1" align="left">EADB</entry><entry namest="col2" nameend="col2" align="left">Ethyl 4-azidohenyl-1,4-dithiobutyrimidate · HCl</entry></row><row><entry namest="col1" nameend="col1" align="left">FNPA</entry><entry namest="col2" nameend="col2" align="left">4-fluoro-3-nitrophenylazide</entry></row><row><entry namest="col1" nameend="col1" align="left">HSAB</entry><entry namest="col2" nameend="col2" align="left">N-hydroxysuccinimidyl 4-azidobenzoate</entry></row><row><entry namest="col1" nameend="col1" align="left">MABI</entry><entry namest="col2" nameend="col2" align="left">Methyl 4-azidobenzoimidate · HCl</entry></row><row><entry namest="col1" nameend="col1" align="left">MBS</entry><entry namest="col2" nameend="col2" align="left">m-maleimidobenzoyl-N-hydroxysuccinimide ester</entry></row><row><entry namest="col1" nameend="col1" align="left">NHS-ASA</entry><entry namest="col2" nameend="col2" align="left">N-hydroxysuccinimidyl-4-azidosalicylic acid</entry></row><row><entry namest="col1" nameend="col1" align="left">MHS</entry><entry namest="col2" nameend="col2" align="left">Maleimidohexanoyl-N-hydroxysuccinimide ester</entry></row><row><entry namest="col1" nameend="col1" align="left">PNP-DTP</entry><entry namest="col2" nameend="col2" align="left">p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate</entry></row><row><entry namest="col1" nameend="col1" align="left">SADP</entry><entry namest="col2" nameend="col2" align="left">N-succinimidyl (4-azidophenyl) 1,3'-dithiopropionate</entry></row><row><entry namest="col1" nameend="col1" align="left">SAND</entry><entry namest="col2" nameend="col2" align="left">Sulfosuccinimidyl 2- (m-azido-o-nitrobenzamido) ethyl 1,3'-dithiopropionate</entry></row><row><entry namest="col1" nameend="col1" align="left">SANPAH</entry><entry namest="col2" nameend="col2" align="left">N-succinimidyl-6 (4'-azido-2'-nitrophenyl-amino) hexanoate</entry></row><row><entry namest="col1" nameend="col1" align="left">SASD</entry><entry namest="col2" nameend="col2" align="left">Sulfosuccinimidyl 2- (p-azidosalicylamido) ethyl 1,3'-dithiopropionate</entry></row><row><entry namest="col1" nameend="col1" align="left">SIAB</entry><entry namest="col2" nameend="col2" align="left">N-succinimidyl (4-iodoacetyl) aminobenzoate</entry></row><row><entry namest="col1" nameend="col1" align="left">SMCC</entry><entry namest="col2" nameend="col2" align="left">Succinimidyl 4- (N-maleimidoethyl) cyclohexane-1-carboxylate</entry></row><row><entry namest="col1" nameend="col1" align="left">SMPB</entry><entry namest="col2" nameend="col2" align="left">Succinimidyl 4- (p-maleimidophenyl) butyrate</entry></row><row><entry namest="col1" nameend="col1" align="left">DSS</entry><entry namest="col2" nameend="col2" align="left">Disuccinimidyl suberate</entry></row><row><entry namest="col1" nameend="col1" align="left">DMS</entry><entry namest="col2" nameend="col2" align="left">Dimethylsuberimidate</entry></row><row><entry namest="col1" nameend="col1" align="left">Traut's reagent</entry><entry namest="col2" nameend="col2" align="left">2-iminothiolane 2,4,6 trichloro-s-triazine</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">SAMBA</entry><entry namest="col2" nameend="col2" align="left">S'-acetyl-mercaptosuccinic anhydride</entry></row></tbody></tgroup></table></tables>

0037To carry out the crosslinking, a solution of the partner P₂ can be mixed with the linker molecules under conditions which lead directly to the crosslinking. The degree of crosslinking in this case is controlled by the amount of linker added.

0038In a further preferred embodiment, the binding partner P₂ is crosslinked with suitable bindable components which are inert with respect to P₁ and the complex to be determined. Suitable for this purpose is, for example, a soluble protein as defined above, in particular bovine serum albumin or human serum albumin.

0039The heterogeneous crosslinking can be carried out, for example, in such a way that both the protein material used as the "inert component" and the specifically bindable partner P₂ are provided with an activated bindable group and are then reacted. In this way, a crosslinked polymer is obtained which contains a sufficient number of bindable partners P₂. Of course, the binding of the partner P₂ to the protein must take place in such a way that neither the specific binding ability with the partner P₁ is impaired, nor the specific binding site for the complex to be determined is blocked.

0040In a further preferred embodiment of the method according to the invention, the binding partner P₂ is crosslinked with other components which have specific binding sites for the receptor. This embodiment is preferably used when P₂ has only one specific binding site for binding with P₁. The other component then provides the binding site for the receptor to be bound. Substances which have specific binding sites, in particular partners of a specific binding pair, as defined above, are suitable as another component.

0041There are various variants for coating the insoluble support material with the polymers I and II. In one embodiment, the insoluble support material is first coated with the polymer I by incubating the support material with a solution which contains polymer I. If polymer I is bound, a solution containing polymer II is then added, the crosslinking of the two polymers then taking place through the specific binding of P₁ with P₂.

0042In another embodiment of the process according to the invention, the insoluble carrier material is incubated with a solution which contains both polymer I and polymer II and, in addition, an inhibitor which inhibits the binding of P₁ to P₂. An inhibitor is used which has a reversible effect and loses its inhibitory effect by simple removal or chemical modification. In the presence of the inhibitor, the partners P₁ and P₂ can be distributed completely homogeneously and, because of the very slow onset of binding, a completely uniform covering of the carrier material with the desired binding-active substance is obtained, which leads to completely uniform and reproducible results even on an industrial scale. This embodiment is suitable if the binding pair P₁-P₂ antigen or hapten antibody or biotin streptavidin or Avidin can be used. As inhibitors of the binding of P₁ and P₂ preferably those substances are used in the context of this embodiment of the invention, which are used in chromosorptive purifications as a desorbent. Acids, bases or chaotropic ions of the Hofmeister series (lyotropic series), as described for example in "Structure and Stability of Biological Macromolecules", 1969, Marcel Dekker, Inc., New York, page 427, and certain, are particularly preferred for this purpose organic compounds or solvents such as acetonitrile, urea or glycerin are used. Both volatile and non-volatile acids can be considered as suitable acids. Volatile acids can be easily removed to remove the inhibitory effect, for example by heating, vacuum and the like. In the case of non-volatile acids, an analogous effect can be achieved by using a salt of a volatile acid, which is decomposed by the non-volatile acid, with the liberation of the volatile acid or by buffering. Preferred examples are acetic acid, propionic acid and hydrochloric acid for volatile acids.

0043Volatile and non-volatile bases such as ammonia and t-phosphate can also be used. Also suitable as inhibitors are organic compounds which can reversibly influence both the protein and the water structure and, for example, in "JF Brandts, Conformial Transitions of Proteins in Water", contained in "Structure and Stability of Biological Macromolecules, 1969, Marcel Dekker, Inc., New York, pages 213-290. Glycerol and urea are particularly preferably used.

0044Chaotropic ions such as thiocyanates and iodides are also suitable as inhibitors. Fluorides, bromides, perchlorates, guanidine and sulfates are also suitable. They can be removed to remove the inhibition of binding by extraction, for example with organic solvents or mixtures of organic solvents or mixtures of organic solvents and water, for example water / alcohol mixtures, optionally with the addition of ionophores and the like. As a rule, a change in the ionic strength is sufficient to achieve the desired effect, but the inhibitor can also be completely removed. The addition of complexing agents such as EDTA is also possible, for example to remove inhibitory metal salts such as MgCl₂.

0045If biotin and streptavidin or avidin are used as the binding pair for polymer I and II, an acid is particularly preferably used as the inhibitor. The strong bond between these two partners can be removed by lowering the pH to values ​​in the range of 4 and below. A volatile acid is preferably used for this, so that the binding effect is then achieved when the volatile acid is released.

0046The two specifically binding partners P₁ and P₂ and optionally the other component are preferably used in such a ratio that P₂ and optionally the other component for the binding of the receptor are present in large excess over their binding partners. In this way, a large number of binding sites are created for the complex to be immobilized. If a partner of the specific binding pair used is already natively present in free form in the sample liquid to be analyzed, it is particularly preferred to provide it with a high binding capacity in order to rule out a disturbance. If, for example, biotin and streptavidin or avidin are used as the specifically binding pair, it is particularly preferred to provide a very high binding capacity for the biotin. Biotin is present in body fluids and can falsify analytical values ​​from determinations in which biotin conjugates are used, especially if, for example, after taking biotin, the serum values ​​are greatly increased. In this case, it is particularly preferred to produce a solid phase matrix with a very high binding capacity for biotin. This is achieved with the method according to the invention, it being possible to create a binding capacity of 200 ng / ml and more for biotin.

0047The solid phase matrix produced according to the invention is used in determinations based on the principle of the immunoassay. It is suitable for the variants of the sandwich test as well as for the variants of the competitive test. There are many variants for these provisions. For example, the sample containing the substance to be determined can be reacted with a receptor that bears a label and at least one other receptor to which a substance that is specifically bindable with the polymer of the specific binding partner P₂ is reacted. The complex to be determined, preferably one of the bound receptors, therefore has a site that is capable of binding to the polymer. This binding site can be identical to or different from that of P₁. This reaction can already take place in a tube coated with the matrix according to the invention or in a correspondingly coated microtiter plate. However, it is also possible to add the solid phase matrix, for example in the form of spheres, only after the incubation. Upon contact with the solid phase matrix, the complex of the substance to be determined, the labeled receptor and the receptor conjugated with the specific binding substance binds to the polymer, which in turn is bound to the carrier via P 1 and the protein. In this way the complex to be determined can be immobilized.

0048Another object of the invention is a solid phase matrix, which is characterized in that it consists of an insoluble carrier material on which a first polymer I, which consists of a large number of partners P₁ of a specific binding pair and a water-soluble biological polymer or derivative thereof with a molecular weight of more than about 20,000, is bound with a second polymer II, which consists only of a large number of the other partner P₂ of the specific binding pair or of P₂, which are crosslinked with other components, the polymer II having both binding sites for P₁ and for an immunologically detectable complex, and wherein the polymers I and II via the specific binding of P₁ are cross-linked with P₂.

0049A solid phase matrix in which the protein is a conjugate of a soluble protein with a molecular weight of 200,000 to 20,000,000 and a large number of biotin, avidin or streptavidin molecules is particularly suitable for carrying out immunoassays. A solid phase matrix is ​​furthermore preferably used, in which the polymer II consists of biotin, avidin or streptavidin molecules. Polymer II is preferably formed from biotin, avidin or streptavidin molecules on the one hand and a hydrophobized protein on the other.

0050According to the invention, there is provided a universal matrix and a method for its production, which can be used in all known immunoassays.

0051This solid phase matrix is ​​independent of the type of receptors used. It is also characterized by high stability.

0052The invention is illustrated by figures and examples.<dl id="dl0001"><dt>Fig. 1</dt><dd>shows two embodiments of the solid phase matrix according to the invention.<ul id="ul0002" list-style="none"><li>a) shows an embodiment of the solid phase matrix according to the invention. On a solid phase 1, a conjugate of a protein 3 with a partner of a specific binding pair P₁ 5 is adsorbed. A polymer 7 is bound to 5, which consists of the other partner P₂ of the specific binding pair. The polymer consists of homogeneously cross-linked identical molecules. Antibodies 9 which are conjugated to P 1 bind to this polymer when carrying out an immunoassay.</li><li>b) shows a further embodiment of the solid phase matrix according to the invention. Here is a solid phase 1, a conjugate of a protein 3 and a partner of a specific binding pair P₁ 5 adsorbed. A polymer 7 is bound to 5. This polymer consists of the other partner of the specific binding pair P₂ and a receptor R. When an immunoassay is carried out, antibodies 9 bind to this polymer, which are conjugated to a substance capable of binding to the receptor R.</li></ul></dd><dt>Fig. 2</dt><dd>shows a diagram in which calibration curves for different coated tubes were set up. The individual curves were obtained with the following tubes:</dd><dt>X:</dt><dd>Luran tubes coated with a two-component matrix consisting of Thermo-RSA-Biotin and homogeneously cross-linked streptavidin.</dd><dt>Triangle:</dt><dd>Luran tubes coated with a two-component matrix consisting of Thermo-RSA-Biotin and Thermo-RSA-Streptavidin</dd><dt>+:</dt><dd>Polystyrene tubes, gamma-irradiated and coated with RSA streptavidin.</dd><dt>Rhombus:</dt><dd>Polystyrene tubes, gamma-irradiated and coated with homogeneously cross-linked streptavidin.</dd><dt>Square:</dt><dd>Luran tubes coated with homogeneously cross-linked streptavidin.</dd><dt>Fig. 3</dt><dd>shows an embodiment of the solid phase matrix according to the invention.</dd></dl>

0053In this embodiment, a first component 3, which has a large number of partners of a specific binding pair, is adsorptively bound to a carrier material 1. A second component 5, which contains a large number of the other partners of the specific binding system, is bound to the first component 3 via the specific bonds 7 of the two partners, with crosslinked polymers of considerable size being formed on the carrier material 1.

example 1

1a) Production of thermal bovine serum albumin biotin

00541 g bovine serum albumin (RSA) is dissolved in 50 ml of 50 mM potassium phosphate (pH 7.8). 1.9 ml of D-biotinyl-ε-aminocaproic acid-N-hydroxysuccinimide ester (NHS-X-biotin, Boehringer Mannheim GmbH) in dimethyl sulfoxide (DMSO) (20 mg / ml) are added dropwise with stirring. The mixture is then incubated at 25 ° C. for 3 hours. After the reaction, dialysis is carried out overnight at 4 ° C. against 50 times the volume of 20 mM potassium phosphate (pH 7.0). The retentate is mixed with the same volume of 20 mM potassium phosphate / 200 mM sodium chloride (pH 7.0), heated to 70 ° C. and incubated at this temperature with careful stirring for 4 hours. The solution is then cooled to room temperature and filtered. The filtrate is dialyzed overnight at 4 ° C. against 50 times the volume of 2 mM potassium phosphate (pH 7.0) and then lyophilized. The product obtained is adsorbed on the solid phase and represents the partner P 1 bound to the soluble protein in the matrix according to the invention.

1b) Activation of streptavidin with maleimido-hexanoyl-N-hydroxysuccinimide ester

005530th mg of streptavidin are dissolved in 3 ml of 30 mM potassium phosphate / 100 mM sodium chloride (pH 7.1) and heated to 25 ° C. 0.15 ml of maleinimido-hexanoyl-N-hydroxysuccinimide ester (MHS) (Boehringer Mannheim GmbH) in DMSO (10 mg / ml) is added dropwise with stirring. After a reaction time of 1 hour at 25 ° C, the solution is cooled in an ice bath. The mixture is then dialyzed twice at 4 ° C. against 1 liter of 50 mM potassium phosphate / 100 mM sodium chloride (pH 5.0).

1c) Activation of streptavidin with S-acetylmercapto-succinic anhydride

005630th mg of streptavidin are dissolved in 3 ml of 100 mM potassium phosphate (pH 7.8) and heated to 25 °. 0.175 ml of S-acetylmercapto-succinic anhydride (SAMBA) in DMSO (10 mg / ml) are added dropwise with stirring. After a reaction time of 3 hours at 25 ° C., dialysis is carried out twice against 1 liter of 50 mM potassium phosphate / 2 mM EDTA (pH 6.5) at 4 ° C.

1d) Homogeneous cross-linking of streptavidin

00573rd ml of a solution of activated SAMBA-streptavidin (10 mg / ml) (preparation according to Example 1c)) are heated to 25 ° and 50 μl of 1M hydroxylamine (pH 6.5) are added. After 30 minutes at 25 ° C., the mixture is diluted by adding 15 ml of 50 mM potassium phosphate / 100 mM sodium chloride / 1 mM EDTA (pH 6.5). The homogeneous crosslinking of the streptavidin is started by adding 3 ml of activated MHS-streptavidin (10 mg / ml) (preparation according to Example 1b)). After a reaction time of 2 hours at 25 ° C. with careful stirring, the reaction is stopped by adding 0.2 ml of 100 mM cysteine ​​/ HCl. After an incubation period of 30 minutes at 25 ° C., the pH of the solution is adjusted to 7.5 by adding 1 M dipotassium hydrogen phosphate. After adding 0.2 ml of 500 mM iodoacetamide, the mixture is incubated at 25 ° C. for a further hour. The mixture is then dialyzed twice against 3 l of 50 mM potassium phosphate / 100 mM sodium chloride (pH 7.5) at 4 ° C. After dialysis, the conjugate is concentrated in an ultrafiltration cell.

0058The homogeneously cross-linked streptavidin can be used either directly or after gel filtration (Superose 6 prep, grade, Pharmacia Uppsala) and renewed concentration for adsorption on the solid phase. It is polymer II in the matrix according to the invention.

Example 2

0059Heterogeneously crosslinked streptavidin is produced which is used as polymer II in the matrix according to the invention.

a) Production of activated SAMBA thermal RSA

0060Thermo-RSA is produced as described under Example 1 a), but here the biotinylation is omitted.

006168 mg of Thermo-RSA are dissolved in 2 ml of 0.1 M potassium phosphate (pH 7.8) and 0.38 ml of SAMBA (10 mg / ml in DMSO) are slowly added. After a reaction time of 3.5 hours at 25 ° C., dialysis is carried out twice against 1 liter of 50 mM potassium phosphate (pH 6.5) at 4 ° C.

b) Preparation of the thermo-RSA-streptavidin conjugate

0062The heterogeneous crosslinking of streptavidin with Thermo-RSA takes place analogously to the homogeneous crosslinking described in Example 1d). 60 mg activated MHS-streptavidin (preparation according to Example 1b)) are reacted with 68 mg activated SAMBA-Thermo-RSA (see above). The reaction product is purified by gel filtration (Superose 6 prep, grade) and concentrated in an ultrafiltration cell. The product obtained is then lyophilized. The product can be used as Polymer II.

Example 3

Loading of Luran® (polystyrene-acrylonitrile copolymer) or γ-irradiated polystyrene tubes

0063The products obtained according to Example 1 and Example 2 are dissolved in 50 mM potassium phosphate (pH 7.4) to a concentration of 10 µg / ml. Then 1.5 ml of a solution of the thermo-RSA-biotin conjugate prepared according to Example 1a) is filled into each tube to be loaded and first loaded for 3 to 5 hours. After complete suction, 1.5 ml of a solution of homogeneously crosslinked streptavidin according to Example 1 or given heterogeneously crosslinked streptavidin according to Example 2 and incubated overnight at room temperature. The tubes are then completely emptied and used for the corresponding tests.

0064For comparison, tubes are only loaded with streptavidin crosslinked according to Example 1 or a conjugate of (non-thermally aggregated) RSA and streptavidin (preparation analogous to Example 2 from activated SAMBA-RSA and activated MHS streptavidin), without pre-loading with biotinylated polymer I.

Example 4

0065The binding capacity of the tubes produced according to Example 3 is determined.

0066The tubes loaded with the different streptavidin polymers according to the invention and the comparison tubes are washed with 1 ml of a solution of biotinylated peroxidase from horseradish (Biotin-POD, Sigma) (10 mU / ml in 50 mM potassium phosphate / 0.5% bovine serum albumin (pH 7.4) ) incubated for 45 minutes at room temperature. The tubes are then emptied and washed twice with bidistilled water. The detection reaction is then carried out using ABTS® (ammonium salt of 2,2'-azino-di (3-ethylbenzothiazoline-6-sulfonic acid)) for 30 minutes at room temperature. The measurement is carried out photometrically at 405 nm. The binding capacity (bika) is determined via a displacement curve. To this end, increasing concentrations (0 to 15 or 0 to 200 ng / ml) of D-biotin (Sigma) are added to the biotin-POD solution. The binding capacity is then calculated from the half-maximum extinction curve obtained by plotting the individual values.

Example 5

0067The stability of the surface adhesion of the matrix coated with protein and biotin and in each case a streptavidin polymer is determined by incubating the loaded tubes with 1.5 ml of a detachment buffer containing detergent (0.2% Tween® 20 in 50 mM potassium phosphate (pH 7.0) ) checked. After an incubation period of one hour at room temperature, 1 ml each is transferred from the tubes to a tube coated with Thermo-RSA-Biotin (preparation according to Example 1a) to determine the amount of conjugate detached. In parallel, to determine a calibration curve, Thermo-RSA-Biotin tubes are mixed with 1 ml release buffer, which contains increasing concentrations of streptavidin. After an incubation period of one hour at room temperature, the tubes are completely emptied and 1 ml of a biotin-POD solution (100 mU / ml in 50 mM potassium phosphate (pH 7.0) is added. After a further incubation of 30 minutes at room temperature the tubes are emptied and then washed three times with bidistilled water. The amount of bound biotin-POD is proportional to the amount of conjugate detached from the tube wall and is determined photometrically by the substrate reaction with ABTS (incubation for one hour at room temperature). Based on the calibration curve, the amount of detached conjugate is quantified and referred to as detached biotin binding capacity.

0068Table 2 shows the biotin binding capacities determined according to Example 4 and the desorption of the conjugates determined according to Example 5 for differently loaded Luran or γ-irradiated polystyrene tubes (PS). The biotin binding capacity (and thus the binding capacity for biotinylated antibodies) of the homogeneously cross-linked specific binding partner directly bound to the solid phase (comparison) (in the example poly-streptavidin) is significantly greater than that of the heterogeneously cross-linked specific binding partner directly bound to the solid phase (comparison ) (in the example RSA-streptavidin, preparation analogous to example 2) from activated SAMBA-RSA and activated MHS-streptavidin). As the removal data show, the cross-linked specific binding partner P₂ can only be applied with a high binding capacity and, if desired, firm wall adhesion if a load with pre-cross-linked protein which contains the specific binding partner P 1, ie biotin covalently bound, is implemented in situ. The influence of the binding capacities and solid-phase detachments of the various conjugates on the sensitivity of a functional test carried out using detergents is described in more detail in Example 6.<tables id="tabl0002" num="0002"><img file="EP0331127B1_D0001.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0331127B1_D0002.tif" /></tables>

Example 6

0069The tubes obtained according to Example 3 are used in a TSH test.

Reagents:

Reagent 1 (antibody incubation solution)

007016 mmol / l phosphate buffer pH 6.9 1.5 µg / ml biotinylated monoclonal antibody against TSH (ECACC 87122201) (The biotinylation was carried out according to JACS 100 (1978), 3585-3590 with biotin by reaction with N-hydroxysuccinimide biotin in a ratio of 10: 1.)

Reagent 2 (antibody-POD conjugate solution)

007136 mmol / l phosphate buffer pH 6.9 2.0 U / ml conjugate of POD and monoclonal antibodies against TSH (ECACC 87122202)

Reagent 3 (substrate chromogen solution)

0072100 mmol / l phosphate citrate buffer pH 4.4 3.2 mmol / l sodium perborate 1.9 mmol / l ABTS® (2,2'-azino-di- [3-ethyl-benzothiazoline-sulfonic acid (6)] - diammonium salt)

0073Tubes which have been coated with various matrices as described in Example 3 are used as the solid phase. 0.2 ml of sample (TSH standard), 0.9 ml of reagent 1 and 0.1 ml of reagent 2 are added to these tubes and incubated for two hours at room temperature. The tubes are then completely emptied and washed three times with water. The POD activity bound to the tube wall is then determined by adding 1 ml of reagent 3 and incubating for one hour by measuring the absorbance at 405 nm. The intensity of the color reaction is proportional to the TSH concentration of the standard. The results are shown in Figure 2.

0074As FIG. 2 shows, the slope of the calibration curve (and thus the sensitivity of the test) increases significantly when using incubation buffers containing detergents from tubes which are loaded with a one-component matrix to tubes which are loaded with the two-component matrix according to the invention . Furthermore, the greatest sensitivity is achieved by using the two-component matrix, which contains as component B a homogeneously crosslinked binding partner (here polymeric streptavidin).

Example 7

0075A matrix is ​​produced in which thermo-RSA is adsorbed onto the solid phase, to which streptavidin is bound as partner P 1. Homogeneously cross-linked, biotinylated protein A is then coupled to the streptavidin as partner P₂.

a) Preparation of thermal RSA streptavidin

0076The preparation is carried out as described in Example 2.

b) Production of homogeneously cross-linked, biotinylated protein A.

007750 mg of Protein A (Boehringer Mannheim GmbH) are dissolved in 5 ml of 30 mM potassium phosphate (pH 7.1) and mixed with a 10-fold molar excess of NHS-X-biotin (dissolved in 10 mg / ml in DMSO). After an incubation period of one hour at 25 ° C., the reaction mixture is dialyzed at 4 ° C. overnight against 10 l of 50 mM potassium phosphate (pH 8.0). The retentate is then concentrated in an ultrafiltration cell to a concentration of 50 mg biotin protein A / ml.

0078The concentrated solution of biotin protein A is heated to 25 ° C. 50 μl of a disuccinimidyl suberate solution (DSS, Pierce; 7 mg / ml in dioxane) are then added with careful stirring. The crosslinking is checked by HPLC on a TSK 3000 gel filtration column (LKB). At intervals of one hour, 50 μl of the DSS solution are added until the peak of monomeric protein A has been reduced to less than 10% of its original size. The further crosslinking is then stopped by adding 50 μl of 1M ethanolamine (pH 8.0). It is incubated overnight at 4 ° C. and then dialyzed twice against 2 l of 2 mM potassium phosphate (pH 7.5). The monomeric protein A is separated off by gel filtration on Superose 12 prep. straight. The homogeneously cross-linked product is collected and concentrated in an ultrafiltration cell. Tubes according to Example 3 are then loaded with these two components.

Example 8

0079It is a matrix consisting of a conjugate of Thermo-RSA with Fcγ fragments of the mouse as P₁ and a homogeneously cross-linked polyclonal anti-mouse Fcγ antibody from sheep as P₂.

a) Preparation of thermal RSA mouse Fcγ fragment

0080The Fcγ fragments are prepared by papain cleavage of mouse immunoglobulins G and separation from the Fab fragments by ion exchange chromatography on DE-52 cellulose by the customary method.

0081Activated MHS-Fcγ fragment is produced analogously to the production of activated MHS streptavidin (Example 1b)). Activated SAMBA-Thermo-RSA is prepared as described in Example 2. The conjugation of 68 mg activated SAMBA-Thermo-RSA with 10 mg activated MHS-Fcγ fragment takes place in the same way as the preparation of heterogeneously cross-linked streptavidin (example 2). The reaction product is purified by gel filtration on Superose 6 prep grade, concentrated in an ultrafiltration cell and then lyophilized.

b) Production of homogeneously cross-linked anti-mouse Fcγ antibody

008250 mg of anti-mouse Fcγ antibodies are dissolved in 1 ml of 50 mM potassium phosphate pH 8.0 and heated to 25 ° C. Analogously to the production of homogeneously crosslinked protein A (example 7b), 50 μl of a DSS solution (7 mg / ml in dioxane) are added at intervals of one hour until the peak in HPLC analysis on a TSK 3000 gel filtration column of the monomeric IgG has dropped to 10% of its original size. Then, as described in Example 7b), the mixture is quenched with ethanolamine and dialyzed. The monomeric IgG is, as described in Example 7b), separated by gel filtration. If necessary, the cross-linked IgG is concentrated by ultrafiltration. Alternatively, the homogeneously crosslinked product can be prepared by activating the antibody with MHS and SAMBA (preparation analogous to Examples 1b) and 1c)) and subsequent crosslinking (analogously to Example 1d)).

0083Tubes according to Example 3 are loaded with these products obtained according to a) and b).

Example 9

0084A matrix consisting of a thermo-RSA-digitoxigenin conjugate as polymer I and homogeneously cross-linked anti-digoxin antibody from sheep as polymer II is produced.

a) Preparation of thermo-RSA digitoxigenin

0085Thermo-RSA is produced as described in Example 1a), but here the biotinylation is omitted. 68 mg of Thermo-RSA are dissolved in 6.8 ml of 50 mM potassium phosphate / 100 mM sodium chloride (pH 8.5) and heated to 25 ° C. 2.86 mg of digitoxigenin-3-succinimidyl-hydroxysuccinimide ester in 0.68 ml of dioxane are added with stirring. After a reaction time of 3 hours at 25 ° C., dialysis is carried out twice against 1 l of 2 mM potassium phosphate (pH 7.2). The reaction product is then concentrated in an ultrafiltration cell.

b) Production of homogeneously cross-linked anti-digoxin antibody

0086The homogeneously cross-linked anti-digoxin antibody is prepared in the same manner as described for the preparation of the homogeneously cross-linked anti-mouse Fcγ antibody (Example 8b)).

0087The tubes are then loaded with solutions of the two products in succession.

0088Digitoxigenin-labeled antibodies are used in the immunoassay. Antibodies labeled in this way are prepared analogously to Example 9a with digitoxigenin-3-succinimidyl-hydroxysuccinimide ester.

Example 10

0089A matrix is ​​produced by incubating the solid phase simultaneously with polymer I and II.

0090A solution consisting of 10 μg / ml poly-streptavidin (preparation according to example 1d) and 0.1 μg / ml thermo-RSA-biotin (preparation according to example 1a) in 5 mmol / l acetic acid is incubated in polystyrene tubes for 20 hours .

0091After removal of the loading solution, 10 mmol / l potassium phosphate buffer, pH 7.2 / 3 g RSA is loaded (30 minutes) and dried after suction.

Example 11

a) Biotinylation of amino-dextran-500

0092100 mg aminodextran, (MW 500,000, 230 NH₂ groups / mol dextran) are dissolved in 5 ml of 100 mM potassium phosphate buffer, pH 8.5. 90 μl of a solution of 10 mg of biotin-N-hydroxysuccinimide ester (Biotin-OSu) in 1 ml of dimethyl sulfoxide (DMSO) are slowly added with stirring.

0093After 2 hours at room temperature, the reaction is stopped with 50 μl 200 mmol / l lysine-HCl, pH 8.5.

0094The mixture is then dialyzed twice against 500 times the volume of 50 mmol / l potassium phosphate buffer, pH 7.2 and diluted with 40 mmol / l potassium phosphate buffer, pH 7.0 to a concentration of 10 µg / ml.

b) loading of polystyrene tubes with amino-dextran-biotin

00951.5 ml portions of this loading solution are filled into polystyrene tubes and incubated at room temperature for 6 hours.

0096It is then suctioned off, 1.5 ml of a homogeneously or heterogeneously crosslinked poly-streptavidin (example 1 or 2) are added and incubated overnight. The tubes are then completely emptied and used for the corresponding tests.

Example 12

0097Thermo-RSA streptavidin tubes, produced according to EP-A 0 269 092, are mixed with 10 µg / ml of Thermo-RSA-Biotin solution (preparation according to Example 1a) in 50 mmol / l potassium phosphate, pH 7.2, for 20 hours incubated.

0098After the solution has been suctioned off completely, 1.5 ml of poly-streptavidin solution (according to Example 1 or 2) are added and incubated overnight at room temperature. The tubes are then completely emptied and used for the corresponding tests.

Example 13

a) Biotinylation of poly (Lys / Phe)

0099100 mg poly (Lys / Phe) HBr (hydrobromide of a copolymer of lysine and phenylalanine) (L-Lys / L-Phe 1: 1, MW 46 KD, manufacturer Sigma) are dissolved in 25 ml of H₂O.

0100180 µl of a solution of 5 mg Biotin-OSu in 1 ml DMSO are slowly added with stirring, the pH being kept constant at 8.0 using an autotitrator.

0101After 2 hours at room temperature, the reaction is stopped with 50 μl 200 mmol / l lysine-HCl, pH 8.5.

0102The mixture is then bidistilled twice against 500 times the volume. H₂O dialyzed and diluted to a concentration of 10 µg / ml.

b) loading of polystyrene tubes with poly (Phe / Lys) biotin

01031.5 ml portions of this loading solution are filled into γ-irradiated polystyrene tubes and incubated at room temperature for 6 hours. It is then suctioned off and 1.5 ml of poly-streptavidin solution (preparation according to Example 1 or 2) are added and incubated at room temperature overnight. The tubes are then completely emptied and used for the corresponding tests.

Example 14

0104A matrix consisting of a Thermo-RSA-biotin conjugate as polymer I and a heterogeneously cross-linked conjugate of streptavidin and anti-digoxin antibodies from sheep as polymer II is produced.

a) Production of Thermo-RSA-Biotin

0105The preparation is carried out as described under Example 1a). The thermo-RSA-biotin conjugate is polymer I in the matrix according to the invention.

b) Preparation of the copolymer from streptavidin and anti-digoxin antibodies from sheep.

010630th mg of streptavidin are activated as described in Example 1c) with S-acetylmercapto-succinic anhydride (SAMBA).

0107At the same time, 30 mg of anti-digoxin antibodies are activated in the same way with maleimido-hexanoyl-N-hydroxysuccinimide ester (MHS), as described in Example 1b) for the MHS activation of streptavidin. A five-fold molar excess of MHS (dissolved in DMSO) compared to the antibodies is used for the reaction.

0108The conjugation of activated SAMBA-streptavidin with activated MHS antibodies takes place in the same way as described for the homogeneous crosslinking of streptavidin in Example 1d).

0109The streptavidin-antibody conjugate obtained in this way can be used either directly or after gel filtration and renewed concentration for adsorption on the solid phase. In the matrix according to the invention, it represents polymer II.

0110To produce the matrix according to the invention, tubes are either coated in succession with a solution of the two products, or at the same time, as described in Example 10, in the presence of 5 mmol / l acetic acid.

0111Digitoxigenin-labeled antibodies are used in the immunoassay. Antibodies labeled in this way are prepared analogously to Example 9a with digitoxigenin-3-succinimidyl-hydroxysuccinimide ester.