CA1335265C - Solid phase matrices and a process for the production thereof - Google Patents
Solid phase matrices and a process for the production thereofInfo
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- CA1335265C CA1335265C CA000592220A CA592220A CA1335265C CA 1335265 C CA1335265 C CA 1335265C CA 000592220 A CA000592220 A CA 000592220A CA 592220 A CA592220 A CA 592220A CA 1335265 C CA1335265 C CA 1335265C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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Abstract
A solid phase matrix comprises an insoluble carrier material to which is bound a protein with a molecular weight of above about 200,000 which has a plurality of components P1 of a specific binding pair and the protein is cross-linked with a polymer of a plurality of the other component P2 of the specific binding pair, the polymer having not only binding points for P1 but also for an immunological complex to be determined; the matrix is useful in determining an analyte in a sample, for example, in an immunoassay; a process for producing the matrix comprises binding the protein to the insoluble carrier and cross-linking with the polymer via a specific binding of P1 with P2.
Description
~ The present invention is concerned with a process ,it for the production of a specifically bindable protein substance bound to an insoluble carrier material, ~9 ; especially for use in a heterogenous analysis process according to the immunoassay principle.
For the determination of a specifically bindable substance, there are frequently used processes according to the immunoassay principle. One of the components of a pair of substances specifically bindable with one another is thereby reacted with the receptor specific for it, which is labelled in known manner. The conjugate of 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 processes. It is thereby advantageous when one of the receptors is present bound to a solid phase. This simplifies the separation of reaction components present bound and non-bound. For the determination of the specifically bindable substance, there is then measured the amount of labelled reaction component bound ~o the solid phase or of the labelled reaction component present in the solution and placed in known manner in relationship to the amount of reaction component to be determined.
As solid phases in the case of the ~immunological processes, there are usually used synthetic resin test .. ~
~' _3_ l 335265 tubes or microtitre plates, on the inner surface of which is fixed the reaction component, or spheres on the outer surface of which is fixed the reaction component. These synthetic resin test tubes, microtitre plates or spheres usually consist of a relatively inert synthetic resin material so that the binding of the reaction component gives rise to difficulties. Further-more, the binding of the specific reaction componen~ to the particular surface must take place in such a way that it does not lose the ability for the specific binding of the substance specifically bindable therewith.
For this reason, the binding of the reaction component to the solid phase is usually adsorptive. Therefore, it has already been suggested to bring about the fixing of the reaction component to the solid phase via a coupling agent which brings about the binding. Care must ~hereby also be taken that the binding of the reaction component to the coupling agent does not destroy the specifically reacting region of the molecule or that the reaction component is bound in such a manner that its reactive position away from the solid phase faces the binding component. Furthermore, it is suggested in Federal Republic of Germany Patent Specification No. 25 33 701, in order to achieve a better binding, to cross-link the individual immunologically active proteins and then to adsorb on polystyrene spheres. A further possibility which is mentioned in l 335265 this literature reference is simultaneously to cross-link an inert protein with the protein with immunological properties so that there results a cross-linked product of inert and active protein which is again adsorbed on to polystyrene spheres. However, depending upon the chosen reaction conditions, this type of cross-linking results in varying and non-reproducible cross-linkings with varying proportions of non-cross-linked as well as insolubilised protein. Furthermore, due to the varying degree of cross-linking, products result with differing binding properties. A similar process is described in European Patent Specification No. 0,122,209 which also displays the same disadvantages. Thus, all of these known processes are still not satisfactory, still do not lead to an optimum adhesion of the specifically bindable substance and are of little suitability for the reproducible production of coated solid phases.
In addition, a further problem is that the anti-body bound to the solid phase must, as a rule, be different for various tests. Therefore, for each test, a specific coated solid phase must be made available, which is very laborious. Since, furthermore, it is not ensured that each antibody immobilised in known manner remains bindable, because non-specific bindings often also take place, and since, furthermore, the dissolving off rate in the case of the previously known processes is very high, the antibody must be used in great excess.
.
.
~5~ 1 335265 .) Nevertheless, the number of binding places is limited.
This results in an increased tendency to disturbance.
~, This is disadvantageous since antibodies are difficult and expensive to produce.
Therefore, it is an object of the present invention to provide a process which reproducibly improves the adhesion of the specifically bindable substance to the solid phase and, furthermore, to provide a process with which a solid phase matrix can be produced which can be used universally for all immunoassays. Since, in the case of many immunological processes, working is carried out with the addition of detergents in order to avoid turbidities and non-specific bindings, it is also an object of the present invention to improve the adhesion to such an extent that, even in the presence of detergents, the bound, specifically bindable substance is not dissolved off.
Thus, according to the present invention, there is provided a process for the production of a specifically bindable protein substance bound to an insoluble carrier material. especially for use in a heterogenous analysis process according to the immuno-assay principle, wherein a protein with a molecular weight of more than about 200,000, which has a plurality of components Pl of a specific binding pair, is first bound to an insoluble carrier material and subsequently cross-linked with a polymer which contains or consists of a plurality of the other component P2 of the specific binding pair via a specific binding of Pl with P2, the polymer thereby having not only binding points for Pl but also for an immunological complex to be determined.
With this process, a solid phase matrix can be produced which can be used for all kinds of immunoassays, for example sandwich tests or competitive tests in a one-step or two-step process. For the sandwich test in the one-step process, the polymer of the component P2 can, for example, provide the binding points on to which the complex to be determined is then immobilised. During the determination process, a sample which contains the substance to be determined is reacted with a labelled receptor, as well as an unlabelled receptor which is bindable with the polymer. The complex formed from the substance to be determined, labelled receptor and receptor with binding points for the polymer then binds, on the basis of the specific bindability, to the polymer so that, in ~his way, the whole complex is immobilised.
After separa~ion of the phases, the labelling can then be measured in one of the two phases.
If the sandwich test is carried out in the two-step process, a solid phase matrix can be used in which an unlabelled receptor is bound in the polymer. The sample and the labelled receptor are then incubated in the presence of this solid phase matrix. After .
separation of the phases, the labelling bound to the solid phase can then be measured.
In the case of carrying out a competitive ~est, sample and labelled sample analogue then compete for an unlabelled receptor. For the test variant of a one-step process, in this case a polymer is used to which the unlabelled receptor is bound. For ~he other variant of the two-step process, the unlabelled receptor is bound during the test in competition with the labelled receptor so that, in this case, a polymer is then used which has binding points for these receptors.
As protein, it is preferred to use one which is more hydrophobic than the components Pl and P2. Soluble proteins are especially preferred with a molecular 15 weight of more than about 200,000 to about 20,000,000 and which are possibly obtained from proteins with a molecular weight of 10,000 to 700,000.
In an especially preferred embodiment, a conjugate is first prepared from a soluble protein 20 with a molecular weight above about 200,000 and a component Pl of a specifically binding pair.
The binding of the component Pl to the protein takes place in known manner, appropriate coupling methods being described, for example, by Ishikawa in 25 J. Immunoassay, 4, 209-327/1983. Either functional groups of the component Pl which are suitable for a binding with the protein are thereby used or, if --8- ~ 335265 . suitable functional groups are not present, these are introduced into the Pl molecule. Thus, for example, in the case of using biotin as Pl, the N-hydroxy-succinimidyl derivative thereof can be bound to the protein by reaction with the amino groups of the protein. Other suitable forms of derivatisation are well known and do not need to be explained here.
Appropriate binding components which can be used as Pl and P2 include, for example, biotin-avidin, biotin-streptavidin, antigen-antibody, hapten-antibody and protein A-immune-~-globulin. By antibodies there are thereby to be understood monoclonal and polyclonal complete antibodies and antibody fragments. If protein A is used as Pl or P2, then in the immunoassay only the receptor which is to be bound to the solid phase should be a complete antibody, whereas as labelled receptor there should be used a Fab or F(ab')2 fragment in order not to bring about a non-specific binding of the labelled receptor with the solid phase which would lead to a falsification of the result.
For the choice of soluble proteins suitable according to the present invention, the molecular weight, as well as the hydrophobicity must be determined in comparison with the corresponding value for the specifically bindable substances. The molecular weight is determined by methods which are known in the art.
A comparison of the hydrophobicity between soluble -9- l 335265 - proteins and specifically bindable substances can also take place according to well-known methods. Appropriate methods include, for example, a comparison of - the fluorescence extinction after binding to coloured material (Biochem. Biophys. Acta, 624, 13-20/1980);
- the elution behaviour in the case of hydrophobic chromatography (Biochem. Biophys. Acta, 576, 269-279/
1979);
- the surface ~ension (Biochem. Biophys. Acta, 670, 64-73/1981);
- the retention times in the case of hydrophobic inter-action chromatography (HIC) (Angew. Chemie, 98, 530-548/1986; J. Chromat., 296, 107-114/1984; Anal.
Biochem., 137, 464-472/1984).
A comparison of the hydrophobicity of substances which are suitable according to the present invention is to be found in Sep. Sci. Technol., 14, 305-317/1979.
According to this, the hydrophobicity increases, for example, in the following sequence:
a2-macroglobulin (M.W. 820,000) bovine serum albumin/human serum albumin (M.W. 70,000) egg albumin a2HS-glycoprotein (M.W. 49,000) ~lc/~lA~glbulin immunoglobulin (M.W. 150,000) transferrin (M.W. 90,000).
Thus, if an immunoglobulin is used as specifically 3 7~
.
-lo- 1 335265 ~- bindable substance, then, for example, human serum albumin or a2HS-glycoprotein are not suitable as soluble proteins in the meaning of the present invention without further pre-treatment.
Both proteins must here be subjected not only to a hydrophobing but also to an increasing of the molecular weight. In the case of transferrin, a cross-linking suffices and in the case of a2-macroglobulin a hydro-phobing is sufficient.
Proteins which are suitable for coupling with immunoglobulin as specific bindable substance without pre-treatment include, for example, ~-lipoproteins (M.W. about 3.2 mio) and a2-lipoprotein (M.W. about 5 to 20 mio).
The hydrophobing can take place, for example by the use of heat, treatment with acids, denaturing agents and/or chaotropic ions and/or by chemical coupling with a hydrophobic compound.
The increasing of the molecular weight can take place, for example, by the use of heat, treatment with acids, denaturing agents and/or chaotropic ions and/or by cross-linking with a bi- or polyfunctional protein reagent.
A protein which is not sufficiently hydrophobic :75 or the molecular weight of which is not sufficiently high is treated until a protein polymer is obtained with a molecular weight of 200,000 or more. It is especially preferred to use a protein polymer which has a molecular weight of 500,000 to 20 mio.
If the protein is to be cross-linked, a hydro-phobing can take place before, during or after the cross-linking. However, the hydrophobing cannot be carriedout in the presence of the specifically bindable sub-stance when the specifically bindable substance is a protein.
For hydrophobing by heating, there is usually employed a temperature of from 40 to 95C. over a period of time of from 1 minute to 10 hours, such as is described, for example, in Biochem. Biophys. Acta, 624, 13-20/1980.
For the treatment with acids, there can be used, for example, acetic acid, propionic acid, lactic acid or hydrochloric acid. Usual concentrations are from 1 to 100 mMole/litre in the case of periods of action of from 10 minutes to 16 hours.
For the treatment with chaotropic ions, there can be used, for example, thiocyanates, iodides, fluorides, bromides, perchlorates and sulphates. As denaturing agents, there can be used, for example, guanidine hydro-chloride or urea. Concentrations of from 10 mMole/litre to 6 mole/litre are here usually employed.
For derivatisation with hydrophobic compounds, there are preferably used soluble fatty acids, lipoids in low or high molecular weight form, as well as -12- ~ 33~5 ` synthetic polymers, such as polypropylene glycol or soluble co-polymers of polystyrene. The derivatisation takes place according to well-known methods.
The cross-linking via bi- or polyfunctional S compounds is carried out with known protein binding agents. These are compounds which mostly carry two functional groups which can be the same or different and which react via these functional groups with functional groups of proteins. Compounds are preferably used which consist of an alkyl chain on the ends of which are present succinimide, maleinimide and/or aldehyde groups.
The protein is then cross-linked in known manner with the bi- or polyfunctional compound by reacting the soluble protein and the bi- or polyfunctional compound.
For the hydrophobing and/or cross-linking, there are preferably used proteins with a molecular weight of from lO,000 to 700,000, bovine serum albumin, lipase or immune-y-globulin being used especially preferably. -The protein or protein conjugate prepared in this way is then bound to an insoluble carrier material. The binding thereby takes place via the protein and is, as a rule, adsorptive. As carrier materials, there can be used the solid phases which are normally employed, for ~5 example Luran, glass, titanium dioxide, polystyrene,Y-activated polystyrene, polystyrene-acrylonitrile co-polymers, paper and/or polyolefins. Before the further * trade-mark -13- l 33~6~
` treatment, the carrier material can be physically or chemically pre-treated. Thus, for example, a synthetic resin surface can be pre-swollen or activated in some other known way. As a rule, the carrier material is present in the form of test tubes, microtitre plates or spheroids but other forms are, however, also possible.
Subsequently, the protein having a plurality of components Pl which is bound to the carrier material is cross-linked with a polymer. This polymer has a plurality of components P2. It can consist either only of P2 or of a mixture of P2 and other components. The polymer has not only binding points for Pl which are provided by P2 but, furthermore, also binding points for an immunological complex to be determined which, in the following, is referred to as receptor. As receptors, there are thereby used specifically bindable substances, especially either specifically bindable complete antibodies which can be polyclonal or mono-clonal, antibody fragments thereof and conjugates of antibodies or antibody fragments with haptens or antigens, as well as haptens and antigens. The binding points of the receptor can either also be provided by P2 or by another component of the polymer.
The individual components P2 can be bound with one another via homo- or hetero-, bi- or polyvalent linkers. The cross-linking is preferably carried out with bivalent linkers since these make possible an .
-14- l 335265 easier control of the degree of polymerisation. How-ever, polyvalent linkers can also be used. As linkers, there can be used those compounds which have reactive groups which are able to react in aqueous solution with the functional groups of the specifically bindable components with the formation of a covalent bond. A
large number of bifunctional and polyfunctional linkers suitable for this purpose is known. Typical examples for homo- or heterobifunctional and trifunctional linkers which are well suited in the scope of the present invention are set out in the following Table 1:
` Table abbreviation chemical designation SPDP N-succinimidyl 3-(2-pyridylthio)-propionate 5 EADB ethyl 4-azidophenyl-1,4-dithiobutyrimid-ate hydrochloride FNPA 4-fluoro-3-nitrophenylazide HSAB N-hydroxysuccinimidyl 4-azidobenzoate MABI methyl 4-azidobenzoimidate hydrochloride 10 MBS -maleimidobenzoyl N-hydroxysuccinimide ester NHS-ASA N-hydroxysuccinimidyl 4-azidosalicylic acid MHS maleimidohexanoyl N-hydroxysuccinimide ester :15 PNP-DTP p-nitrophenyl 2-diazo-3,3,3-trifluoro-propionate SADP N-succinimidyl (4-azidophenyl)-1,3'-dithiopropionate SAND sulphosuccinimidyl 2-(m-azido-o-nitro-benzamido)-ethyl 1,3'-dithiopropionate SANPAH N-succinimidyl 6-(4'-azido-2'-nitro-phenylamino)-hexanoate SASD sulphosuccinimidyl 2-(p-azidosalicyl-amido)-ethyl-1,3'-dithiopropionate 25 SIAB N-succinimidyl (4-iodoacetyl)-amino-benzoate SMCC succinimidyl 4-(N-maleinimidoethyl)-cyclohexane-l-carboxylate SMPB succinimidyl 4-(p-maleimidophenyl)-butyrate DSS disuccinimidyl suberate DMS dimethyl suberimidate Traut's 2-iminothiolane + 2,4,6-trichloro-s-reagent triazine 35 SAMBA s'-acetylmercaptosuccinic anhydride For carrying out the cross-linking, a solution of the component P2 is mixed with the linker molecules under conditions which lead directly to the cross-linking. In this case, the extent of the cross-linking is controlled by the amount of linker added.
In a further preferred embodiment, the binding component P2 is cross-linked with appropriate bindable components which are inert with regard to Pl and to the complex to be determined. For this purpose, there can ]0 be used, for example, a soluble protein such as was defined hereinbefore, especially bovine serum albumin or human serum albumin. ~
The heterogenous cross-linking can, for exarnple, take place in such a manner that not only the protein material used as "inert component" but also the specifically bindable component P2 are provided with an activated bindable group and subsequently reacted. In this way, there is obtained a cross-linked polymer which contains a sufficient number of bindable components P2.
The binding of the component P2 to the protein must, of course, thereby take place in such a manner that neither the specific bindability with the component Pl is impaired nor is the specific binding point for the complex to be determined blocked.
In a further preferred embodiment of the process according to the present invention, the binding component P2 is cross-linked with other components which have specific binding points for the receptor. This embodiment is specifically used when P2 only has one specific binding point for the binding with Pl. The other component then provides the bindin~ point for the receptor to be bound. As other components, there can be used substances which have specific binding points, especially componen~s of a specifically-binding pair such as are defined hereinbefore.
The two specifically binding components Pl and P2 and possibly the other components are preferably used in such a ratio that P2 and possibly the other components are present in large excess in comparison with their binding components. In this way, very many binding points are provided for the complex to be immobilised.
On the other hand, the binding to the carrier material is very stable since, even in the case of the dissolving off of some components Pl from the protein, the binding by the cross-linking of the second component is still present.
~0 The solid phase matrix produced according to the present invention is used for determinations according to the immunoassay principle. It can be used not only for the variants of the sandwich test but also for the variants of the competitive test. There are many variants for these determinations. For example, the sample which contains the substance to be determined can thereby be reacted with a receptor which carries a , labelling and at least one further receptor to which is bound a substance specifically bindable with the polymer of the specifically binding component P2. The complex to be determined, preferably one of the bound receptors, thus has a point which is bindable with the polymer. This binding point can be identical to or different from that of Pl. This reaction can already take place in a test tube coated with the matrix according to the present invention or in an appropriately coated microtitre plate. However, it is also possible to add the solid phase matrix, for example in the form of spheroids, only after the incubation. In the case of contact with the solid phase matrix, the complex of substance to be bound, labelled receptor and receptor conjugated with specifically binding substance then binds to the polymer which, in turn, is bound via Pl and the protein to the carrier. In this way, the complex to be determined can be immobilised.
The present invention also provides a solid phase matrlx, wherein it consists of an insoluble carrier material to which is bound a protein with a molecular weight of over about 200,000 which has a plurality of components Pl of a specific binding pair and the protein is cross-linked with a polymer of a plurality of the other components P2 of the specific binding pair, the polymer having not only binding points for Pl but also for an immunological complex to be determined.
-19- 1 33526~
For carrying out immunoassays, a solid phase matrix is especially preferred in which the protein is a conjugate of a soluble protein with a molecular weight of from 200,000 to 20,000,000 and of a plurality of biotin, avidin or strep~avidin molecules. Further-more, a solid phase matrix is preferably used in which the polymer consists of biotin, avidin or streptavidin molecules. The polymer is preferably formed from biotin, avidin or streptavidin and a hydrophobed protein.
According to the present invention, a universal matrix is provided, as well as a process for the production thereof, which can be used in all known immunoassays.
This solid phase matrix is independent of the nature of the receptors used. Furthermore, it is characterised by a high stability.
The following Examples are given for the purpose of illustrating the present invention, with reference to the accompanying drawings, in which:
Fig. 1 shows two embodiments of the solid phase matrix according to the present invention:
a) shows one embodiment of the solid phase matrix according to the present invention. On to a solid phase 1 is adsorbed a conjugate of a protein 3 with one component 5 of a specific binding pair Pl. On 5 is bound a polymer 7 which consists of the other component P2 of the specific binding pair. The polymer consists of homogeneous cross-linked identical molecules.
In the case of carrying out an immunoassay, antibodies 9, which are conjugated with Pl, bind to this polymer.
b) Shows a further embodiment of the solid phase matrix according to the present invention. Here, on a solid phase 1 is adsorbed a conjugate of a protein 3 and a component 5 of a specific binding pair Pl. On 5 is bound a polymer 7. This polymer consists of the other component of the specific binding pair P2, as well as a receptor R. In the carrying out of an immunoassay, on this polymer bind antibodies 9 which are conjugated with a substance bindable with the receptor R.
Fig. 2 shows a diagram in which are plotted calibration curves for different coated test tubes. The curves of this diagram have the following meanings:
X : Two-component matrix consisting of thermo-BSA-biotin and homogeneously cross-linked streptavidin; Luran test tubes;
: Two-component matrix consisting of thermo-BSA-biotin and thermo-BSA-streptavidin; Luran test tubes + : BSA-streptavidin on y-irradiated polystyrene test tubes 1 33526~
: Homogeneously cross-linked streptavidin on irradiated polystyrene test tubes : Homogeneously cross-linked streptavidin on Luran test tubes.
The individual curves were thereby obtained with the following test tubes:
X : Luran test tubes coated with a two-component matrix consisting of thermo-BSA-biotin and homogeneously cross-linked streptavidin 0 ~: Luran test tubes coated with a two-component matrix consisting of thermo-BSA-biotin and thermo-BSA-streptavidin + : polystyrene test tubes Y-irradiated and coated with BSA-streptavidin :15 0 : polystyrene test tubes ~-irradiated and coated wi~h homogeneously cross-linked streptavidin : Luran test tubes coated with homogeneously cross-linked strep~avidin.
Fig. 3 shows one embodiment of the solid phase matrix according to the present invention. In this embodiment, on a carrier material 1 is adsorpt-ively bound a first component 3 which has a plurality o components of a specific binding pair. A second cornponent 5, which contains a plurality of the other component o the specific binding system, is bound via the specific bindings 7 of the two components to the irst component 3, B
~. ..~
-22- ~ 3 cross-linked polymers of considerable size thereby resulting on the carrier material 1.
Example 1.
la) Production of thermo-bovine serum albumin-biotin.
1 g. Bovine serum albumin (BSA) is dissolved in 50 ml. 50 mM potassium phosphate (pH 7.8). While stirring, 1.9 ml. D-biotinyl-Y-aminocapronic acid N-hydroxysuccinimide ester (NHS-X-biotin; Boehringer Mannheim GmbH) in dimethyl sulphoxide (20 mg./ml.) are added dropwise thereto. Subsequently, the reaction mixture is incubated for 3 hours at 25C. After react-ing, it is dialysed overnight at 4C. against a 50 fold 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 70C. and incubated for 4 hours at this temperature, with careful stirring. Subsequently, the solution is cooled to ambient temperature and filtered.
The filtrate is dialysed overnight at 4C. against a ,70 50 fold volume of 2 mM potassium phosphate (pH 7.0) and subsequently lyophilised. The product obtained is adsorbed on the solid phase and represents, in the matrix according to the present invention, the component Pl bound to the soluble protein.
lb) Activation of streptavidin with maleimidohexanoyl N-hydroxysuccinimide ester.
30 mg. Streptavidin are dissolved in 3 ml. 30 mM
-23- 1 33~65 potassium phosphate/100 mM sodium chloride (pH 7.1) and tempered to 25C. While stirring, 0.15 ml.
maleinimidohexanoyl N-hydroxysuccinimide ester (MHS) (Boehringer Mannheim GmbH) in dimethyl sulphoxide (10 mg./ml.) is added dropwise thereto. After a reaction time of 1 hour at 25C., the solution is cooled in an ice-bath. Subsequently, it is dialysed twice at 4C. against 1 litre 50 mM potassium phosphate/
100 mM sodium chloride (pH 5.0) lc Activation of strepavidin with S-acetylmercapto-succinic anhydride.
30 mg. Strepavidin are dissolved in 3 ml. 100 mM
potassium phosphate (pH 7.8) and tempered to 25C.
While stirring, 0.175 ml. S-acetylmercapto-succinic anhydride (SAMBA) in dimethyl sulphoxide (10 mg./ml.) is added dropwise thereto. After a reaction time of 3 hours at 25C., it is dialysed at 4C. against 1 litre 50 mM potassium phosphate/2 mM ethylenediamine-tetraacetic acid (EDTA) (pH 6.5).
ld) Homogeneous cross-linking of streptavidin.
3 ml. of a solution of activated SAMBA-streptavidin (10 mg./ml.) (preparation according to Example lc)) are tempered to 25C. and mixed with 50 ~1. lM hydroxylamine (pH 6.5). After 30 minutes at 25C., dilution is carried out by the addition of 15 ml. 50 mM potassium phosphate/
100 mM sodium chloride/l mM EDTA (pH 6.5). The homogen-eous cross-linking of the streptavidin is started by the -24- 1 3~
addition of 3 ml. activated MHS-streptavidin (10 mg./ml.) - (preparation according to Example lb)). After a reaction time of 2 hours at 25C. with careful stirring, the reaction is terminated by the addition of 0.2 ml. 100 mM cysteine hydrochloride. After an incubation time of 30 minutes at 25C., the pH value of the solution is adjusted to 7.5 by the addition of lM dipotassium hydrogen phosphate. After the addition of 0.2 ml. 500 mM iodoacetamide, the reaction mixture is incubated for a further hour at 25C. Subsequently, it is dialysed twice at 4C. against 3 litres 50 mM
potassium phosphate/100 mM sodium chloride (pH 7.5).
After the dialysis, the conjugate is concentrated in an ultrafiltration cell.
The homogeneously cross-linked streptavidin can either be used directly or after gel filtration (Superose*6 prep. grade; Pharmacia, Sweden) and renewed concentration for adsorption to the solid phase. In the matrix according to the present invention, it represents the cross-linked component P2.
Example 2.
Thermo-BSA is produced as described in Example la) but here the biotinylation is omitted.
68 mg. Thermo-BSA are dissolved in 2 ml. O.lM
potassium phosphate (pH 7.8) and slowly mixed with 0.38 ml. SAMBA (10 mg./ml. in dimethyl sulphoxide).
After a reaction time of 3.5 hours at 25C., it is * trade-mark -!
-25- ~ 33~65 dialysed twice at 4C. against l litre 50 mM potassium phosphate (pH 6.5).
Heterogeneously cross-linked streptavidin is .! produced for use as P2 in a matrix according to the present invention. The heterogeneous cross-linking of streptavidin with thermo-BSA takes place analogously to the homogeneous cross-linking described in Example ld). 60 mg. activated MHS-streptavidin (production according to Example lb)) are thereby reacted with 68 mg. activated SAMBA-thermo-BSA (see above). The reaction product is purified by gel filtration (Superose 6 prep. grade) and concentrated in an ultra-filtration cell. The product obtained is subsequently lyophilised. The product can be used as P2.
Example 3.
Loading of Luran*(polystyrene-acrylonitrile co-polymer) or of ~-irradiated polystyrene test tubes.
The products obtained according to Examples 1 and 2 are dissolved in 50 mM potassium phosphate (pH 7.4) to give a concentration of lO ~g./ml. Into each test tube to be loaded there is then placed 1.5 ml. of a solution of the thermo-BSA-biotin conjugate produced according to Example la) and first loaded for 3 to 5 hours. Subsequently, after complete sucking out, there is introduced into the test tubes 1.5 ml. of a solution of homogeneously cross-linked streptavidin according to Example 1 or of heterogeneously cross-linked * Trade Mark _~. .
-streptavidin according to Example 2 and incubated overnight at ambient temperature. Thereafter, the test tubes are completely emptied and used for the corresponding tests.
For comparison, test tubes are only loaded with cross-linked streptavidin according to Example 1 or 2 or a conjugate of streptavidin and thermo-BSA obtained according to Example 7, without pre-loading with biotinylated protein.
Example 4.
Determination of the binding capacity of test tubes produced according to Example 3.
The test tubes loaded with the different strept-avidin polymers according to the present invention, as well as the comparison test tubes, are incubated with 1 ml. of a solution of biotinylated horse radish peroxidase (biotin-POD, Sigma) (10 mU/ml. in 50 mM
potassium phosphate/0.5% BSA (pH 7.4)) for 45 minutes at ambient temperature. The test tubes are then emptied and washed twice with double distilled water.
Subsequently, the detection reaction takes place with the use of ABTS ~ (ammonium salt of 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulphonic acid) for 30 minutes at ambient temperature. The measurement is carried out photometrically at 405 nm. The binding capacity (Bica) is determined via a displacement curve. For this purpose, to the biotin-POD solution are added -27~
increasing concentrations (0 to 15 or 0 to 200 ng./ml., respectively) of D-biotin (Sigma). The binding capacity is then calculated from the curves obtained by plotting the individual values from the semi-maximum extinction.
Example 5.
The stabili~y of the surface adhesion of the matrix coated with protein and biotin and, in each case, a streptavidin polymer is tested by incubation of the loaded test tubes with 1.5 ml. of a detergent-containing dissolving-off buffer (0.2% Tween 20 in 50 mM potassium phosphate (pH 7.0)). After an incubation time of 1 hour at ambient temperature, for the determination of the amount of conjugate dissolved off, in each case 1 ml. is transferred from the test tube into a test tube coated with a thermo-BSA-biotin (preparation according to Example la)). Parallel thereto, for the production of a calibration curve, to thermo-BSA-biotin test tubes is added 1 ml. of dissolving-off buffer which contains increasing con-centrations of streptavidin. After an incubation time of 1 hour at ambient temperature, the test tubes are completely emptied and mixed with 1 ml. of a biotin-POD
solution (100 mU/ml. in 50 mM potassium phosphate (pH
7.0)). After a further incubation of 30 minutes at ambient temperature, the tubes are emptied and subse-quently washed three times with double distilled water.
B * Trade Mark .. ... i~
The amount of bound biotin-POD is proportional to the amount of conjugate dissolved off from the test tube wall and is determined photometrically by the ` substrate reaction with ABTS (lncubation for 1 hour at ambient temperature). On the basis of the calibration curve, there is quantified the amount of conjugate dissolved off and designated the dissolved-off biotin binding capacity.
The following Table 2 shows, for various Luran and Y-irradiated polystyrene test tubes, the biotin binding capacities determined according to Example 4 and the desorption of the conjugates determined accord-ing to Example 5. The biotin binding capacity (and thus the binding capacity for biotinylated antibodies) of the homogeneously cross-linked specific binding component bound to the solid phase (comparison) (in this example poly-streptavidin) is distinctly greater than that of the heterogenously cross-linked specific binding component bound to the solid phase (comparison) (in this example BSA-streptavidin; preparation analogously to Example le) from activated SAMBA-BSA and activated MHS-streptavidin). As the dissolving off data show, the cross-linked specific binding component P2 can, however, then only be applied with high binding capacity and desired secure wall adhesion when previously a loading with pre-cross-linked protein, which contains covalently bound the specific binding component Pl, i.e. biotin, is reacted in situ. The influence of the binding capacities and solid phase dissolving off of the various conjugates on the sensitivity of a function test carried out with the use of detergents is des-cribed in more detail in Example 6.
Table 2 coating BSA-SA pSA pSA T-BSA-biotinl T-BSA-biotin (comp- (comp- (comp- + 2 2 +
arison) arison) arison) T-BSA-SA pSA
(according to (according to the the invention) invention) test tube r-PS Y-PS Luran Luran Luran material loading con-centration 10 10 10 10 /82 10 /82 (~g./ml.) L5 biotin-Bica 7.5 58 60 15 120 (ng) desorption (Bi-Bica) 0.017 0.267 9.3 0.005 0.015 (ng) % Bi-Bica 0 3 0 5 15.5 0.03 0.01 Abbreviations:
BSA-SA = bovine serum albumin-streptavidin conjugate pSA = polymeric homogeneously cross-linked streptavidin T-BSA-biotin or -SA = thermo-BSA-biotin or -streptavidin y-PS = irradiated polystyrene test tubes biotin-Bica or Bi-Bica = biotin binding capacity.
Example 6.
The test tubes obtained according to Example 5 are used in a TSH tes~.
Reagents:
Reagent 1 tantibody incubation solution) 16 mMole/litre phosphate buffer (pH 6.9) 1.5 ~g./ml. biotinylated monoclonal antibody aginst TSH (ECACC 87122201) (the biotinylation took place according to J.A.C.S., 100, 3585-3590/1978 with biotin by reaction with N-hydroxysuccinimide-biotin in a ratio of 10:1).
Reagen~ 2 (antibody-POD conjugate solution) 36 mMole/litre phosphate buffer (pH 6.9) 2.0 U/ml. conjugate of POD and monoclonal anti-bodies against TSH (ECACC 87122202).
Reagent 3 (substrate chromogen solution) 100 mMole/litre phosphate-citrate buffer (pH 4.4) 3.2 mMole/litre sodium perborate 1.9 mMole/litre ABTS (diammonium salt of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid).
As solid phases, there are used test tubes which have been coated with different matrices as described in Example 3. In these test tubes are placed 0.2 ml. of sample (TSH standard), 0.9 ml. Reagent 1 and 0.1 ml.
Reagent 2 and incubated for 2 hours at ambient ~ 335265 temperature. Subsequently, the test tubes are com-pletely emptied and washed three times with water. The POD activity bound to the test tube walls is then determined, a~ter adding l ml. of ~eagent 3 and incubating for 1 hour, by measuring the extinction at 405 nm. The intensity of the colour reaction is proportional to the TSH concentration of the standard.
The results obtained are shown in Fig. 2 of the accompanying drawings.
As Fig. 2 shows, the gradient of the calibration curve (and thus the sensitivity of the test) increases distinctly in the case of using detergent-containing incubation buffer froril test tubes which are loaded with a single-component matrix to test tubes which are loaded with the two-co;nponent matrix according to the present invention. Furthermore, the greatest sensitivity is achieved by the us~ of the two-corllponent matrix which, as component B, contains a homogeneously cross-linked binding component (here polymaric strept-avidin).Example 7.
A matrix is produced in which, on the solid phase, thermo-BSA is adsorbed to which streptavidin is bound as component Pl. On to the streptavidin is then coupled hornogeneously cross-linked, biotinylated protein A as component P2.
-32- l 335265 a) Preparation of thermo-BSA streptavidin.
.~ The preparation takes place as described in ~xample le).
b) Preparation of llomogeneously cross-linked, biotinylated protein A.
50 mg. protein A (Boehringer Mannheim GmbH) are dissolved in 5 ml. 30 mM potassium pllosphate (pH 7.1) and mixed with a lO fold molar excess of NHS-X-biotin (dissolved in an amount of 10 mg./ml. in dimethyl sulphoxide). After an incubation period of l hour at 25C., the reaction mixture is dialysed overnight at 4C. against 10 litres 50 mM potassiurn pnosphate (pH
8.0). The retentate is subsequently concentrated in an ultrafiltration cell to a concentration of 50 mg.
biotin-protein A/ml.
The concentrated solution of biotin-protein A is warmed to 25C. Subsequently, while carefully stirring, 50 ~1. of a disuccinimidyl suberate solution (DSS, Pierce; 7 mg./ml. dioxan) are added thereto. The cross-linking is monitored by IIPLC on a TSK 3000 gel filtration column (LKB). At intervals of 1 hour, in each case 50 ~1. of the DSS solution are added thereto until the peak of the monomeric protein A has been reduced to less than 10% of its initial size. There-after, the further cross-linking is stopped by the addition of 50 ~1. lM ethanolamine (pH 8.0). Incub-ation is carried out overnight at 4C. and subsequently dialysis is carried out twice against 2 litres 2mM
~, potassium phosphate (pH 7.5). The separation of the monomeric protein A takes place by gel filtration on ..
Superose 12 prep. grade. The homogeneously cross-linked product is collected and concentrated in an ultrafiltration cell. Test tubes are then loaded according to Exa,-nple 3 with these two components.
Example 8.
A matrix is prepared consisting of a conjugate of thermo-BSA with mouse F ~fragments as Pl and a homogeneously cross-linked polyclonal anti-mouse-Fc antibody from sheep as P2.
a) Preparation of thermo-BSA-mouse Fc~fragment.
The Fc~fragments are prepared by papain cleavage of r.louse immunoglobulins G and separation of the Fab fragments by ion exchange chromatography on DE-52-cellulose according to conventional processes.
Activated MHS-Fc~fragment is prepared analogously to the preparation of activated MHS-streptavidin (see Example lb)). Activated SAMBA-thermo-BSA is prepared as described in Example le). The conjugate of 6~ mg.
activated SAMBA-thermo-BSA with 10 mg. activated MHS-Fc~
fragment takes plac2 in the same way as the preparation of heterogeneously cross-linked streptavidin (see Example le)). The reaction product is purified by gel filtration on Superose 6 prep. grade, concentrated in an ultrafiltration cell and subsequently lyophilised.
b) Preparation of homogeneously cross-linked anti-, mouse-Fc~antibodies.
50 mg. Anti-mouse-Fc~antibodies are dissolved in 1 ml. 50 mM potassium phosphate (pII 8.0) and warmed to 25C. Analogously to the preparation of homogeneously cross-linked protein A (see Example 2b)), at intervals of 1 hour there are, in eacl~ case~ added 50 ~1. of a DSS solution (7 Mg./ml. in dioxan) until in the HPLC
analysis on a TSK 3000 gel filtration column, the peak of the ~ono~eric IgGs has dropped to 10% o~ its initial size. Subsequently, as described in Example 7b ), the reaction is stopped with ethanolamine and then dialysed.
The monomeric IgG is, as described in Example 7b), separated off by gel filtration. The cross-linked IgG
is possibly concentrated by ultrafiltration. Altern-atively, the hornogeneously cross-linked product can be prepared by activation of the antibody witl~ MHS and SAiviBA (preparation analogously to Examples lb) and lc)) and subsequent cross-linking (analogously to Example ld)).
Test tubes are loaded according to Exa.nple 3 with these products obtained according to a) and b).
Exa~ple 9.
A matrix is prepared consisting of a thermo-~SA-digitoxigenin conjugate as Pl and homogeneously cross-linked anti-digoxin-antibody from sheep as P2.
* trade-mark . .
a) Preparation of thermo-BSA-digitoxigenin.
Thermo-BSA is prepared as described in Example la).
68 mg. Thermo-BSA are dissolved in 6.8 ml. 50 m~i , potassium phosphate/100 mM sodiurn chloride (pH 8.5) and warmed ~o 25C. I~hile stirring, 2.86 rng. digitoxigenin-3-succinimidyl hydroxysuccinimide ester in 0.68 ml.
dioxan are added thereto. After a reaction time of 3 hours at 25C., dialysis is carried out twice against 1 litre 2 mM potassium phosphate (pH 7.2). Subsequently, the reaction product is concentrated in an ultra-filtration cell.
b) Preparation of a homogeneously cross-linked anti-digoxin antibody.
The preparation of the homogeneously cross-linked anti-digoxin antibody takes place in the same way as described for the preparation of the homogeneously cross-linked anti--.nouse-Fc~antibody (Example 8b)).
Subsequently, test tubes are successively loaded with solutions of the two products.
Digitoxigenin-labelled antibodies are used in the immunoassay. The preparation of such labelled anti-bodies takes place analogously to Example 9a) with digitoxigenin-3-succinimidyl hydroxysuccinir.lide ester.
The Patent Specifications referred to herein are more fully identified below.
Federal Republic of Germany Patent Specification (Offenlegungsschrift) 2533701, filed July 28, 1975, published (laid open) February 12, 1976, W.F. Barg, assigned to American Cyanamid Co .
European Patent Specification 0,122,209, L0 published October 17, 1984, M. Delaage et al, assigned to Immunotech S.A.
For the determination of a specifically bindable substance, there are frequently used processes according to the immunoassay principle. One of the components of a pair of substances specifically bindable with one another is thereby reacted with the receptor specific for it, which is labelled in known manner. The conjugate of 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 processes. It is thereby advantageous when one of the receptors is present bound to a solid phase. This simplifies the separation of reaction components present bound and non-bound. For the determination of the specifically bindable substance, there is then measured the amount of labelled reaction component bound ~o the solid phase or of the labelled reaction component present in the solution and placed in known manner in relationship to the amount of reaction component to be determined.
As solid phases in the case of the ~immunological processes, there are usually used synthetic resin test .. ~
~' _3_ l 335265 tubes or microtitre plates, on the inner surface of which is fixed the reaction component, or spheres on the outer surface of which is fixed the reaction component. These synthetic resin test tubes, microtitre plates or spheres usually consist of a relatively inert synthetic resin material so that the binding of the reaction component gives rise to difficulties. Further-more, the binding of the specific reaction componen~ to the particular surface must take place in such a way that it does not lose the ability for the specific binding of the substance specifically bindable therewith.
For this reason, the binding of the reaction component to the solid phase is usually adsorptive. Therefore, it has already been suggested to bring about the fixing of the reaction component to the solid phase via a coupling agent which brings about the binding. Care must ~hereby also be taken that the binding of the reaction component to the coupling agent does not destroy the specifically reacting region of the molecule or that the reaction component is bound in such a manner that its reactive position away from the solid phase faces the binding component. Furthermore, it is suggested in Federal Republic of Germany Patent Specification No. 25 33 701, in order to achieve a better binding, to cross-link the individual immunologically active proteins and then to adsorb on polystyrene spheres. A further possibility which is mentioned in l 335265 this literature reference is simultaneously to cross-link an inert protein with the protein with immunological properties so that there results a cross-linked product of inert and active protein which is again adsorbed on to polystyrene spheres. However, depending upon the chosen reaction conditions, this type of cross-linking results in varying and non-reproducible cross-linkings with varying proportions of non-cross-linked as well as insolubilised protein. Furthermore, due to the varying degree of cross-linking, products result with differing binding properties. A similar process is described in European Patent Specification No. 0,122,209 which also displays the same disadvantages. Thus, all of these known processes are still not satisfactory, still do not lead to an optimum adhesion of the specifically bindable substance and are of little suitability for the reproducible production of coated solid phases.
In addition, a further problem is that the anti-body bound to the solid phase must, as a rule, be different for various tests. Therefore, for each test, a specific coated solid phase must be made available, which is very laborious. Since, furthermore, it is not ensured that each antibody immobilised in known manner remains bindable, because non-specific bindings often also take place, and since, furthermore, the dissolving off rate in the case of the previously known processes is very high, the antibody must be used in great excess.
.
.
~5~ 1 335265 .) Nevertheless, the number of binding places is limited.
This results in an increased tendency to disturbance.
~, This is disadvantageous since antibodies are difficult and expensive to produce.
Therefore, it is an object of the present invention to provide a process which reproducibly improves the adhesion of the specifically bindable substance to the solid phase and, furthermore, to provide a process with which a solid phase matrix can be produced which can be used universally for all immunoassays. Since, in the case of many immunological processes, working is carried out with the addition of detergents in order to avoid turbidities and non-specific bindings, it is also an object of the present invention to improve the adhesion to such an extent that, even in the presence of detergents, the bound, specifically bindable substance is not dissolved off.
Thus, according to the present invention, there is provided a process for the production of a specifically bindable protein substance bound to an insoluble carrier material. especially for use in a heterogenous analysis process according to the immuno-assay principle, wherein a protein with a molecular weight of more than about 200,000, which has a plurality of components Pl of a specific binding pair, is first bound to an insoluble carrier material and subsequently cross-linked with a polymer which contains or consists of a plurality of the other component P2 of the specific binding pair via a specific binding of Pl with P2, the polymer thereby having not only binding points for Pl but also for an immunological complex to be determined.
With this process, a solid phase matrix can be produced which can be used for all kinds of immunoassays, for example sandwich tests or competitive tests in a one-step or two-step process. For the sandwich test in the one-step process, the polymer of the component P2 can, for example, provide the binding points on to which the complex to be determined is then immobilised. During the determination process, a sample which contains the substance to be determined is reacted with a labelled receptor, as well as an unlabelled receptor which is bindable with the polymer. The complex formed from the substance to be determined, labelled receptor and receptor with binding points for the polymer then binds, on the basis of the specific bindability, to the polymer so that, in ~his way, the whole complex is immobilised.
After separa~ion of the phases, the labelling can then be measured in one of the two phases.
If the sandwich test is carried out in the two-step process, a solid phase matrix can be used in which an unlabelled receptor is bound in the polymer. The sample and the labelled receptor are then incubated in the presence of this solid phase matrix. After .
separation of the phases, the labelling bound to the solid phase can then be measured.
In the case of carrying out a competitive ~est, sample and labelled sample analogue then compete for an unlabelled receptor. For the test variant of a one-step process, in this case a polymer is used to which the unlabelled receptor is bound. For ~he other variant of the two-step process, the unlabelled receptor is bound during the test in competition with the labelled receptor so that, in this case, a polymer is then used which has binding points for these receptors.
As protein, it is preferred to use one which is more hydrophobic than the components Pl and P2. Soluble proteins are especially preferred with a molecular 15 weight of more than about 200,000 to about 20,000,000 and which are possibly obtained from proteins with a molecular weight of 10,000 to 700,000.
In an especially preferred embodiment, a conjugate is first prepared from a soluble protein 20 with a molecular weight above about 200,000 and a component Pl of a specifically binding pair.
The binding of the component Pl to the protein takes place in known manner, appropriate coupling methods being described, for example, by Ishikawa in 25 J. Immunoassay, 4, 209-327/1983. Either functional groups of the component Pl which are suitable for a binding with the protein are thereby used or, if --8- ~ 335265 . suitable functional groups are not present, these are introduced into the Pl molecule. Thus, for example, in the case of using biotin as Pl, the N-hydroxy-succinimidyl derivative thereof can be bound to the protein by reaction with the amino groups of the protein. Other suitable forms of derivatisation are well known and do not need to be explained here.
Appropriate binding components which can be used as Pl and P2 include, for example, biotin-avidin, biotin-streptavidin, antigen-antibody, hapten-antibody and protein A-immune-~-globulin. By antibodies there are thereby to be understood monoclonal and polyclonal complete antibodies and antibody fragments. If protein A is used as Pl or P2, then in the immunoassay only the receptor which is to be bound to the solid phase should be a complete antibody, whereas as labelled receptor there should be used a Fab or F(ab')2 fragment in order not to bring about a non-specific binding of the labelled receptor with the solid phase which would lead to a falsification of the result.
For the choice of soluble proteins suitable according to the present invention, the molecular weight, as well as the hydrophobicity must be determined in comparison with the corresponding value for the specifically bindable substances. The molecular weight is determined by methods which are known in the art.
A comparison of the hydrophobicity between soluble -9- l 335265 - proteins and specifically bindable substances can also take place according to well-known methods. Appropriate methods include, for example, a comparison of - the fluorescence extinction after binding to coloured material (Biochem. Biophys. Acta, 624, 13-20/1980);
- the elution behaviour in the case of hydrophobic chromatography (Biochem. Biophys. Acta, 576, 269-279/
1979);
- the surface ~ension (Biochem. Biophys. Acta, 670, 64-73/1981);
- the retention times in the case of hydrophobic inter-action chromatography (HIC) (Angew. Chemie, 98, 530-548/1986; J. Chromat., 296, 107-114/1984; Anal.
Biochem., 137, 464-472/1984).
A comparison of the hydrophobicity of substances which are suitable according to the present invention is to be found in Sep. Sci. Technol., 14, 305-317/1979.
According to this, the hydrophobicity increases, for example, in the following sequence:
a2-macroglobulin (M.W. 820,000) bovine serum albumin/human serum albumin (M.W. 70,000) egg albumin a2HS-glycoprotein (M.W. 49,000) ~lc/~lA~glbulin immunoglobulin (M.W. 150,000) transferrin (M.W. 90,000).
Thus, if an immunoglobulin is used as specifically 3 7~
.
-lo- 1 335265 ~- bindable substance, then, for example, human serum albumin or a2HS-glycoprotein are not suitable as soluble proteins in the meaning of the present invention without further pre-treatment.
Both proteins must here be subjected not only to a hydrophobing but also to an increasing of the molecular weight. In the case of transferrin, a cross-linking suffices and in the case of a2-macroglobulin a hydro-phobing is sufficient.
Proteins which are suitable for coupling with immunoglobulin as specific bindable substance without pre-treatment include, for example, ~-lipoproteins (M.W. about 3.2 mio) and a2-lipoprotein (M.W. about 5 to 20 mio).
The hydrophobing can take place, for example by the use of heat, treatment with acids, denaturing agents and/or chaotropic ions and/or by chemical coupling with a hydrophobic compound.
The increasing of the molecular weight can take place, for example, by the use of heat, treatment with acids, denaturing agents and/or chaotropic ions and/or by cross-linking with a bi- or polyfunctional protein reagent.
A protein which is not sufficiently hydrophobic :75 or the molecular weight of which is not sufficiently high is treated until a protein polymer is obtained with a molecular weight of 200,000 or more. It is especially preferred to use a protein polymer which has a molecular weight of 500,000 to 20 mio.
If the protein is to be cross-linked, a hydro-phobing can take place before, during or after the cross-linking. However, the hydrophobing cannot be carriedout in the presence of the specifically bindable sub-stance when the specifically bindable substance is a protein.
For hydrophobing by heating, there is usually employed a temperature of from 40 to 95C. over a period of time of from 1 minute to 10 hours, such as is described, for example, in Biochem. Biophys. Acta, 624, 13-20/1980.
For the treatment with acids, there can be used, for example, acetic acid, propionic acid, lactic acid or hydrochloric acid. Usual concentrations are from 1 to 100 mMole/litre in the case of periods of action of from 10 minutes to 16 hours.
For the treatment with chaotropic ions, there can be used, for example, thiocyanates, iodides, fluorides, bromides, perchlorates and sulphates. As denaturing agents, there can be used, for example, guanidine hydro-chloride or urea. Concentrations of from 10 mMole/litre to 6 mole/litre are here usually employed.
For derivatisation with hydrophobic compounds, there are preferably used soluble fatty acids, lipoids in low or high molecular weight form, as well as -12- ~ 33~5 ` synthetic polymers, such as polypropylene glycol or soluble co-polymers of polystyrene. The derivatisation takes place according to well-known methods.
The cross-linking via bi- or polyfunctional S compounds is carried out with known protein binding agents. These are compounds which mostly carry two functional groups which can be the same or different and which react via these functional groups with functional groups of proteins. Compounds are preferably used which consist of an alkyl chain on the ends of which are present succinimide, maleinimide and/or aldehyde groups.
The protein is then cross-linked in known manner with the bi- or polyfunctional compound by reacting the soluble protein and the bi- or polyfunctional compound.
For the hydrophobing and/or cross-linking, there are preferably used proteins with a molecular weight of from lO,000 to 700,000, bovine serum albumin, lipase or immune-y-globulin being used especially preferably. -The protein or protein conjugate prepared in this way is then bound to an insoluble carrier material. The binding thereby takes place via the protein and is, as a rule, adsorptive. As carrier materials, there can be used the solid phases which are normally employed, for ~5 example Luran, glass, titanium dioxide, polystyrene,Y-activated polystyrene, polystyrene-acrylonitrile co-polymers, paper and/or polyolefins. Before the further * trade-mark -13- l 33~6~
` treatment, the carrier material can be physically or chemically pre-treated. Thus, for example, a synthetic resin surface can be pre-swollen or activated in some other known way. As a rule, the carrier material is present in the form of test tubes, microtitre plates or spheroids but other forms are, however, also possible.
Subsequently, the protein having a plurality of components Pl which is bound to the carrier material is cross-linked with a polymer. This polymer has a plurality of components P2. It can consist either only of P2 or of a mixture of P2 and other components. The polymer has not only binding points for Pl which are provided by P2 but, furthermore, also binding points for an immunological complex to be determined which, in the following, is referred to as receptor. As receptors, there are thereby used specifically bindable substances, especially either specifically bindable complete antibodies which can be polyclonal or mono-clonal, antibody fragments thereof and conjugates of antibodies or antibody fragments with haptens or antigens, as well as haptens and antigens. The binding points of the receptor can either also be provided by P2 or by another component of the polymer.
The individual components P2 can be bound with one another via homo- or hetero-, bi- or polyvalent linkers. The cross-linking is preferably carried out with bivalent linkers since these make possible an .
-14- l 335265 easier control of the degree of polymerisation. How-ever, polyvalent linkers can also be used. As linkers, there can be used those compounds which have reactive groups which are able to react in aqueous solution with the functional groups of the specifically bindable components with the formation of a covalent bond. A
large number of bifunctional and polyfunctional linkers suitable for this purpose is known. Typical examples for homo- or heterobifunctional and trifunctional linkers which are well suited in the scope of the present invention are set out in the following Table 1:
` Table abbreviation chemical designation SPDP N-succinimidyl 3-(2-pyridylthio)-propionate 5 EADB ethyl 4-azidophenyl-1,4-dithiobutyrimid-ate hydrochloride FNPA 4-fluoro-3-nitrophenylazide HSAB N-hydroxysuccinimidyl 4-azidobenzoate MABI methyl 4-azidobenzoimidate hydrochloride 10 MBS -maleimidobenzoyl N-hydroxysuccinimide ester NHS-ASA N-hydroxysuccinimidyl 4-azidosalicylic acid MHS maleimidohexanoyl N-hydroxysuccinimide ester :15 PNP-DTP p-nitrophenyl 2-diazo-3,3,3-trifluoro-propionate SADP N-succinimidyl (4-azidophenyl)-1,3'-dithiopropionate SAND sulphosuccinimidyl 2-(m-azido-o-nitro-benzamido)-ethyl 1,3'-dithiopropionate SANPAH N-succinimidyl 6-(4'-azido-2'-nitro-phenylamino)-hexanoate SASD sulphosuccinimidyl 2-(p-azidosalicyl-amido)-ethyl-1,3'-dithiopropionate 25 SIAB N-succinimidyl (4-iodoacetyl)-amino-benzoate SMCC succinimidyl 4-(N-maleinimidoethyl)-cyclohexane-l-carboxylate SMPB succinimidyl 4-(p-maleimidophenyl)-butyrate DSS disuccinimidyl suberate DMS dimethyl suberimidate Traut's 2-iminothiolane + 2,4,6-trichloro-s-reagent triazine 35 SAMBA s'-acetylmercaptosuccinic anhydride For carrying out the cross-linking, a solution of the component P2 is mixed with the linker molecules under conditions which lead directly to the cross-linking. In this case, the extent of the cross-linking is controlled by the amount of linker added.
In a further preferred embodiment, the binding component P2 is cross-linked with appropriate bindable components which are inert with regard to Pl and to the complex to be determined. For this purpose, there can ]0 be used, for example, a soluble protein such as was defined hereinbefore, especially bovine serum albumin or human serum albumin. ~
The heterogenous cross-linking can, for exarnple, take place in such a manner that not only the protein material used as "inert component" but also the specifically bindable component P2 are provided with an activated bindable group and subsequently reacted. In this way, there is obtained a cross-linked polymer which contains a sufficient number of bindable components P2.
The binding of the component P2 to the protein must, of course, thereby take place in such a manner that neither the specific bindability with the component Pl is impaired nor is the specific binding point for the complex to be determined blocked.
In a further preferred embodiment of the process according to the present invention, the binding component P2 is cross-linked with other components which have specific binding points for the receptor. This embodiment is specifically used when P2 only has one specific binding point for the binding with Pl. The other component then provides the bindin~ point for the receptor to be bound. As other components, there can be used substances which have specific binding points, especially componen~s of a specifically-binding pair such as are defined hereinbefore.
The two specifically binding components Pl and P2 and possibly the other components are preferably used in such a ratio that P2 and possibly the other components are present in large excess in comparison with their binding components. In this way, very many binding points are provided for the complex to be immobilised.
On the other hand, the binding to the carrier material is very stable since, even in the case of the dissolving off of some components Pl from the protein, the binding by the cross-linking of the second component is still present.
~0 The solid phase matrix produced according to the present invention is used for determinations according to the immunoassay principle. It can be used not only for the variants of the sandwich test but also for the variants of the competitive test. There are many variants for these determinations. For example, the sample which contains the substance to be determined can thereby be reacted with a receptor which carries a , labelling and at least one further receptor to which is bound a substance specifically bindable with the polymer of the specifically binding component P2. The complex to be determined, preferably one of the bound receptors, thus has a point which is bindable with the polymer. This binding point can be identical to or different from that of Pl. This reaction can already take place in a test tube coated with the matrix according to the present invention or in an appropriately coated microtitre plate. However, it is also possible to add the solid phase matrix, for example in the form of spheroids, only after the incubation. In the case of contact with the solid phase matrix, the complex of substance to be bound, labelled receptor and receptor conjugated with specifically binding substance then binds to the polymer which, in turn, is bound via Pl and the protein to the carrier. In this way, the complex to be determined can be immobilised.
The present invention also provides a solid phase matrlx, wherein it consists of an insoluble carrier material to which is bound a protein with a molecular weight of over about 200,000 which has a plurality of components Pl of a specific binding pair and the protein is cross-linked with a polymer of a plurality of the other components P2 of the specific binding pair, the polymer having not only binding points for Pl but also for an immunological complex to be determined.
-19- 1 33526~
For carrying out immunoassays, a solid phase matrix is especially preferred in which the protein is a conjugate of a soluble protein with a molecular weight of from 200,000 to 20,000,000 and of a plurality of biotin, avidin or strep~avidin molecules. Further-more, a solid phase matrix is preferably used in which the polymer consists of biotin, avidin or streptavidin molecules. The polymer is preferably formed from biotin, avidin or streptavidin and a hydrophobed protein.
According to the present invention, a universal matrix is provided, as well as a process for the production thereof, which can be used in all known immunoassays.
This solid phase matrix is independent of the nature of the receptors used. Furthermore, it is characterised by a high stability.
The following Examples are given for the purpose of illustrating the present invention, with reference to the accompanying drawings, in which:
Fig. 1 shows two embodiments of the solid phase matrix according to the present invention:
a) shows one embodiment of the solid phase matrix according to the present invention. On to a solid phase 1 is adsorbed a conjugate of a protein 3 with one component 5 of a specific binding pair Pl. On 5 is bound a polymer 7 which consists of the other component P2 of the specific binding pair. The polymer consists of homogeneous cross-linked identical molecules.
In the case of carrying out an immunoassay, antibodies 9, which are conjugated with Pl, bind to this polymer.
b) Shows a further embodiment of the solid phase matrix according to the present invention. Here, on a solid phase 1 is adsorbed a conjugate of a protein 3 and a component 5 of a specific binding pair Pl. On 5 is bound a polymer 7. This polymer consists of the other component of the specific binding pair P2, as well as a receptor R. In the carrying out of an immunoassay, on this polymer bind antibodies 9 which are conjugated with a substance bindable with the receptor R.
Fig. 2 shows a diagram in which are plotted calibration curves for different coated test tubes. The curves of this diagram have the following meanings:
X : Two-component matrix consisting of thermo-BSA-biotin and homogeneously cross-linked streptavidin; Luran test tubes;
: Two-component matrix consisting of thermo-BSA-biotin and thermo-BSA-streptavidin; Luran test tubes + : BSA-streptavidin on y-irradiated polystyrene test tubes 1 33526~
: Homogeneously cross-linked streptavidin on irradiated polystyrene test tubes : Homogeneously cross-linked streptavidin on Luran test tubes.
The individual curves were thereby obtained with the following test tubes:
X : Luran test tubes coated with a two-component matrix consisting of thermo-BSA-biotin and homogeneously cross-linked streptavidin 0 ~: Luran test tubes coated with a two-component matrix consisting of thermo-BSA-biotin and thermo-BSA-streptavidin + : polystyrene test tubes Y-irradiated and coated with BSA-streptavidin :15 0 : polystyrene test tubes ~-irradiated and coated wi~h homogeneously cross-linked streptavidin : Luran test tubes coated with homogeneously cross-linked strep~avidin.
Fig. 3 shows one embodiment of the solid phase matrix according to the present invention. In this embodiment, on a carrier material 1 is adsorpt-ively bound a first component 3 which has a plurality o components of a specific binding pair. A second cornponent 5, which contains a plurality of the other component o the specific binding system, is bound via the specific bindings 7 of the two components to the irst component 3, B
~. ..~
-22- ~ 3 cross-linked polymers of considerable size thereby resulting on the carrier material 1.
Example 1.
la) Production of thermo-bovine serum albumin-biotin.
1 g. Bovine serum albumin (BSA) is dissolved in 50 ml. 50 mM potassium phosphate (pH 7.8). While stirring, 1.9 ml. D-biotinyl-Y-aminocapronic acid N-hydroxysuccinimide ester (NHS-X-biotin; Boehringer Mannheim GmbH) in dimethyl sulphoxide (20 mg./ml.) are added dropwise thereto. Subsequently, the reaction mixture is incubated for 3 hours at 25C. After react-ing, it is dialysed overnight at 4C. against a 50 fold 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 70C. and incubated for 4 hours at this temperature, with careful stirring. Subsequently, the solution is cooled to ambient temperature and filtered.
The filtrate is dialysed overnight at 4C. against a ,70 50 fold volume of 2 mM potassium phosphate (pH 7.0) and subsequently lyophilised. The product obtained is adsorbed on the solid phase and represents, in the matrix according to the present invention, the component Pl bound to the soluble protein.
lb) Activation of streptavidin with maleimidohexanoyl N-hydroxysuccinimide ester.
30 mg. Streptavidin are dissolved in 3 ml. 30 mM
-23- 1 33~65 potassium phosphate/100 mM sodium chloride (pH 7.1) and tempered to 25C. While stirring, 0.15 ml.
maleinimidohexanoyl N-hydroxysuccinimide ester (MHS) (Boehringer Mannheim GmbH) in dimethyl sulphoxide (10 mg./ml.) is added dropwise thereto. After a reaction time of 1 hour at 25C., the solution is cooled in an ice-bath. Subsequently, it is dialysed twice at 4C. against 1 litre 50 mM potassium phosphate/
100 mM sodium chloride (pH 5.0) lc Activation of strepavidin with S-acetylmercapto-succinic anhydride.
30 mg. Strepavidin are dissolved in 3 ml. 100 mM
potassium phosphate (pH 7.8) and tempered to 25C.
While stirring, 0.175 ml. S-acetylmercapto-succinic anhydride (SAMBA) in dimethyl sulphoxide (10 mg./ml.) is added dropwise thereto. After a reaction time of 3 hours at 25C., it is dialysed at 4C. against 1 litre 50 mM potassium phosphate/2 mM ethylenediamine-tetraacetic acid (EDTA) (pH 6.5).
ld) Homogeneous cross-linking of streptavidin.
3 ml. of a solution of activated SAMBA-streptavidin (10 mg./ml.) (preparation according to Example lc)) are tempered to 25C. and mixed with 50 ~1. lM hydroxylamine (pH 6.5). After 30 minutes at 25C., dilution is carried out by the addition of 15 ml. 50 mM potassium phosphate/
100 mM sodium chloride/l mM EDTA (pH 6.5). The homogen-eous cross-linking of the streptavidin is started by the -24- 1 3~
addition of 3 ml. activated MHS-streptavidin (10 mg./ml.) - (preparation according to Example lb)). After a reaction time of 2 hours at 25C. with careful stirring, the reaction is terminated by the addition of 0.2 ml. 100 mM cysteine hydrochloride. After an incubation time of 30 minutes at 25C., the pH value of the solution is adjusted to 7.5 by the addition of lM dipotassium hydrogen phosphate. After the addition of 0.2 ml. 500 mM iodoacetamide, the reaction mixture is incubated for a further hour at 25C. Subsequently, it is dialysed twice at 4C. against 3 litres 50 mM
potassium phosphate/100 mM sodium chloride (pH 7.5).
After the dialysis, the conjugate is concentrated in an ultrafiltration cell.
The homogeneously cross-linked streptavidin can either be used directly or after gel filtration (Superose*6 prep. grade; Pharmacia, Sweden) and renewed concentration for adsorption to the solid phase. In the matrix according to the present invention, it represents the cross-linked component P2.
Example 2.
Thermo-BSA is produced as described in Example la) but here the biotinylation is omitted.
68 mg. Thermo-BSA are dissolved in 2 ml. O.lM
potassium phosphate (pH 7.8) and slowly mixed with 0.38 ml. SAMBA (10 mg./ml. in dimethyl sulphoxide).
After a reaction time of 3.5 hours at 25C., it is * trade-mark -!
-25- ~ 33~65 dialysed twice at 4C. against l litre 50 mM potassium phosphate (pH 6.5).
Heterogeneously cross-linked streptavidin is .! produced for use as P2 in a matrix according to the present invention. The heterogeneous cross-linking of streptavidin with thermo-BSA takes place analogously to the homogeneous cross-linking described in Example ld). 60 mg. activated MHS-streptavidin (production according to Example lb)) are thereby reacted with 68 mg. activated SAMBA-thermo-BSA (see above). The reaction product is purified by gel filtration (Superose 6 prep. grade) and concentrated in an ultra-filtration cell. The product obtained is subsequently lyophilised. The product can be used as P2.
Example 3.
Loading of Luran*(polystyrene-acrylonitrile co-polymer) or of ~-irradiated polystyrene test tubes.
The products obtained according to Examples 1 and 2 are dissolved in 50 mM potassium phosphate (pH 7.4) to give a concentration of lO ~g./ml. Into each test tube to be loaded there is then placed 1.5 ml. of a solution of the thermo-BSA-biotin conjugate produced according to Example la) and first loaded for 3 to 5 hours. Subsequently, after complete sucking out, there is introduced into the test tubes 1.5 ml. of a solution of homogeneously cross-linked streptavidin according to Example 1 or of heterogeneously cross-linked * Trade Mark _~. .
-streptavidin according to Example 2 and incubated overnight at ambient temperature. Thereafter, the test tubes are completely emptied and used for the corresponding tests.
For comparison, test tubes are only loaded with cross-linked streptavidin according to Example 1 or 2 or a conjugate of streptavidin and thermo-BSA obtained according to Example 7, without pre-loading with biotinylated protein.
Example 4.
Determination of the binding capacity of test tubes produced according to Example 3.
The test tubes loaded with the different strept-avidin polymers according to the present invention, as well as the comparison test tubes, are incubated with 1 ml. of a solution of biotinylated horse radish peroxidase (biotin-POD, Sigma) (10 mU/ml. in 50 mM
potassium phosphate/0.5% BSA (pH 7.4)) for 45 minutes at ambient temperature. The test tubes are then emptied and washed twice with double distilled water.
Subsequently, the detection reaction takes place with the use of ABTS ~ (ammonium salt of 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulphonic acid) for 30 minutes at ambient temperature. The measurement is carried out photometrically at 405 nm. The binding capacity (Bica) is determined via a displacement curve. For this purpose, to the biotin-POD solution are added -27~
increasing concentrations (0 to 15 or 0 to 200 ng./ml., respectively) of D-biotin (Sigma). The binding capacity is then calculated from the curves obtained by plotting the individual values from the semi-maximum extinction.
Example 5.
The stabili~y of the surface adhesion of the matrix coated with protein and biotin and, in each case, a streptavidin polymer is tested by incubation of the loaded test tubes with 1.5 ml. of a detergent-containing dissolving-off buffer (0.2% Tween 20 in 50 mM potassium phosphate (pH 7.0)). After an incubation time of 1 hour at ambient temperature, for the determination of the amount of conjugate dissolved off, in each case 1 ml. is transferred from the test tube into a test tube coated with a thermo-BSA-biotin (preparation according to Example la)). Parallel thereto, for the production of a calibration curve, to thermo-BSA-biotin test tubes is added 1 ml. of dissolving-off buffer which contains increasing con-centrations of streptavidin. After an incubation time of 1 hour at ambient temperature, the test tubes are completely emptied and mixed with 1 ml. of a biotin-POD
solution (100 mU/ml. in 50 mM potassium phosphate (pH
7.0)). After a further incubation of 30 minutes at ambient temperature, the tubes are emptied and subse-quently washed three times with double distilled water.
B * Trade Mark .. ... i~
The amount of bound biotin-POD is proportional to the amount of conjugate dissolved off from the test tube wall and is determined photometrically by the ` substrate reaction with ABTS (lncubation for 1 hour at ambient temperature). On the basis of the calibration curve, there is quantified the amount of conjugate dissolved off and designated the dissolved-off biotin binding capacity.
The following Table 2 shows, for various Luran and Y-irradiated polystyrene test tubes, the biotin binding capacities determined according to Example 4 and the desorption of the conjugates determined accord-ing to Example 5. The biotin binding capacity (and thus the binding capacity for biotinylated antibodies) of the homogeneously cross-linked specific binding component bound to the solid phase (comparison) (in this example poly-streptavidin) is distinctly greater than that of the heterogenously cross-linked specific binding component bound to the solid phase (comparison) (in this example BSA-streptavidin; preparation analogously to Example le) from activated SAMBA-BSA and activated MHS-streptavidin). As the dissolving off data show, the cross-linked specific binding component P2 can, however, then only be applied with high binding capacity and desired secure wall adhesion when previously a loading with pre-cross-linked protein, which contains covalently bound the specific binding component Pl, i.e. biotin, is reacted in situ. The influence of the binding capacities and solid phase dissolving off of the various conjugates on the sensitivity of a function test carried out with the use of detergents is des-cribed in more detail in Example 6.
Table 2 coating BSA-SA pSA pSA T-BSA-biotinl T-BSA-biotin (comp- (comp- (comp- + 2 2 +
arison) arison) arison) T-BSA-SA pSA
(according to (according to the the invention) invention) test tube r-PS Y-PS Luran Luran Luran material loading con-centration 10 10 10 10 /82 10 /82 (~g./ml.) L5 biotin-Bica 7.5 58 60 15 120 (ng) desorption (Bi-Bica) 0.017 0.267 9.3 0.005 0.015 (ng) % Bi-Bica 0 3 0 5 15.5 0.03 0.01 Abbreviations:
BSA-SA = bovine serum albumin-streptavidin conjugate pSA = polymeric homogeneously cross-linked streptavidin T-BSA-biotin or -SA = thermo-BSA-biotin or -streptavidin y-PS = irradiated polystyrene test tubes biotin-Bica or Bi-Bica = biotin binding capacity.
Example 6.
The test tubes obtained according to Example 5 are used in a TSH tes~.
Reagents:
Reagent 1 tantibody incubation solution) 16 mMole/litre phosphate buffer (pH 6.9) 1.5 ~g./ml. biotinylated monoclonal antibody aginst TSH (ECACC 87122201) (the biotinylation took place according to J.A.C.S., 100, 3585-3590/1978 with biotin by reaction with N-hydroxysuccinimide-biotin in a ratio of 10:1).
Reagen~ 2 (antibody-POD conjugate solution) 36 mMole/litre phosphate buffer (pH 6.9) 2.0 U/ml. conjugate of POD and monoclonal anti-bodies against TSH (ECACC 87122202).
Reagent 3 (substrate chromogen solution) 100 mMole/litre phosphate-citrate buffer (pH 4.4) 3.2 mMole/litre sodium perborate 1.9 mMole/litre ABTS (diammonium salt of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid).
As solid phases, there are used test tubes which have been coated with different matrices as described in Example 3. In these test tubes are placed 0.2 ml. of sample (TSH standard), 0.9 ml. Reagent 1 and 0.1 ml.
Reagent 2 and incubated for 2 hours at ambient ~ 335265 temperature. Subsequently, the test tubes are com-pletely emptied and washed three times with water. The POD activity bound to the test tube walls is then determined, a~ter adding l ml. of ~eagent 3 and incubating for 1 hour, by measuring the extinction at 405 nm. The intensity of the colour reaction is proportional to the TSH concentration of the standard.
The results obtained are shown in Fig. 2 of the accompanying drawings.
As Fig. 2 shows, the gradient of the calibration curve (and thus the sensitivity of the test) increases distinctly in the case of using detergent-containing incubation buffer froril test tubes which are loaded with a single-component matrix to test tubes which are loaded with the two-co;nponent matrix according to the present invention. Furthermore, the greatest sensitivity is achieved by the us~ of the two-corllponent matrix which, as component B, contains a homogeneously cross-linked binding component (here polymaric strept-avidin).Example 7.
A matrix is produced in which, on the solid phase, thermo-BSA is adsorbed to which streptavidin is bound as component Pl. On to the streptavidin is then coupled hornogeneously cross-linked, biotinylated protein A as component P2.
-32- l 335265 a) Preparation of thermo-BSA streptavidin.
.~ The preparation takes place as described in ~xample le).
b) Preparation of llomogeneously cross-linked, biotinylated protein A.
50 mg. protein A (Boehringer Mannheim GmbH) are dissolved in 5 ml. 30 mM potassium pllosphate (pH 7.1) and mixed with a lO fold molar excess of NHS-X-biotin (dissolved in an amount of 10 mg./ml. in dimethyl sulphoxide). After an incubation period of l hour at 25C., the reaction mixture is dialysed overnight at 4C. against 10 litres 50 mM potassiurn pnosphate (pH
8.0). The retentate is subsequently concentrated in an ultrafiltration cell to a concentration of 50 mg.
biotin-protein A/ml.
The concentrated solution of biotin-protein A is warmed to 25C. Subsequently, while carefully stirring, 50 ~1. of a disuccinimidyl suberate solution (DSS, Pierce; 7 mg./ml. dioxan) are added thereto. The cross-linking is monitored by IIPLC on a TSK 3000 gel filtration column (LKB). At intervals of 1 hour, in each case 50 ~1. of the DSS solution are added thereto until the peak of the monomeric protein A has been reduced to less than 10% of its initial size. There-after, the further cross-linking is stopped by the addition of 50 ~1. lM ethanolamine (pH 8.0). Incub-ation is carried out overnight at 4C. and subsequently dialysis is carried out twice against 2 litres 2mM
~, potassium phosphate (pH 7.5). The separation of the monomeric protein A takes place by gel filtration on ..
Superose 12 prep. grade. The homogeneously cross-linked product is collected and concentrated in an ultrafiltration cell. Test tubes are then loaded according to Exa,-nple 3 with these two components.
Example 8.
A matrix is prepared consisting of a conjugate of thermo-BSA with mouse F ~fragments as Pl and a homogeneously cross-linked polyclonal anti-mouse-Fc antibody from sheep as P2.
a) Preparation of thermo-BSA-mouse Fc~fragment.
The Fc~fragments are prepared by papain cleavage of r.louse immunoglobulins G and separation of the Fab fragments by ion exchange chromatography on DE-52-cellulose according to conventional processes.
Activated MHS-Fc~fragment is prepared analogously to the preparation of activated MHS-streptavidin (see Example lb)). Activated SAMBA-thermo-BSA is prepared as described in Example le). The conjugate of 6~ mg.
activated SAMBA-thermo-BSA with 10 mg. activated MHS-Fc~
fragment takes plac2 in the same way as the preparation of heterogeneously cross-linked streptavidin (see Example le)). The reaction product is purified by gel filtration on Superose 6 prep. grade, concentrated in an ultrafiltration cell and subsequently lyophilised.
b) Preparation of homogeneously cross-linked anti-, mouse-Fc~antibodies.
50 mg. Anti-mouse-Fc~antibodies are dissolved in 1 ml. 50 mM potassium phosphate (pII 8.0) and warmed to 25C. Analogously to the preparation of homogeneously cross-linked protein A (see Example 2b)), at intervals of 1 hour there are, in eacl~ case~ added 50 ~1. of a DSS solution (7 Mg./ml. in dioxan) until in the HPLC
analysis on a TSK 3000 gel filtration column, the peak of the ~ono~eric IgGs has dropped to 10% o~ its initial size. Subsequently, as described in Example 7b ), the reaction is stopped with ethanolamine and then dialysed.
The monomeric IgG is, as described in Example 7b), separated off by gel filtration. The cross-linked IgG
is possibly concentrated by ultrafiltration. Altern-atively, the hornogeneously cross-linked product can be prepared by activation of the antibody witl~ MHS and SAiviBA (preparation analogously to Examples lb) and lc)) and subsequent cross-linking (analogously to Example ld)).
Test tubes are loaded according to Exa.nple 3 with these products obtained according to a) and b).
Exa~ple 9.
A matrix is prepared consisting of a thermo-~SA-digitoxigenin conjugate as Pl and homogeneously cross-linked anti-digoxin-antibody from sheep as P2.
* trade-mark . .
a) Preparation of thermo-BSA-digitoxigenin.
Thermo-BSA is prepared as described in Example la).
68 mg. Thermo-BSA are dissolved in 6.8 ml. 50 m~i , potassium phosphate/100 mM sodiurn chloride (pH 8.5) and warmed ~o 25C. I~hile stirring, 2.86 rng. digitoxigenin-3-succinimidyl hydroxysuccinimide ester in 0.68 ml.
dioxan are added thereto. After a reaction time of 3 hours at 25C., dialysis is carried out twice against 1 litre 2 mM potassium phosphate (pH 7.2). Subsequently, the reaction product is concentrated in an ultra-filtration cell.
b) Preparation of a homogeneously cross-linked anti-digoxin antibody.
The preparation of the homogeneously cross-linked anti-digoxin antibody takes place in the same way as described for the preparation of the homogeneously cross-linked anti--.nouse-Fc~antibody (Example 8b)).
Subsequently, test tubes are successively loaded with solutions of the two products.
Digitoxigenin-labelled antibodies are used in the immunoassay. The preparation of such labelled anti-bodies takes place analogously to Example 9a) with digitoxigenin-3-succinimidyl hydroxysuccinir.lide ester.
The Patent Specifications referred to herein are more fully identified below.
Federal Republic of Germany Patent Specification (Offenlegungsschrift) 2533701, filed July 28, 1975, published (laid open) February 12, 1976, W.F. Barg, assigned to American Cyanamid Co .
European Patent Specification 0,122,209, L0 published October 17, 1984, M. Delaage et al, assigned to Immunotech S.A.
Claims (18)
1. A process for the production of a solid phase matrix of an insoluble carrier with a specifically bindable substance bound thereto, comprising:
(i) coating a substance on an insoluble carrier material, said substance containing a first polymer, said first polymer being a protein polymer having a molecular weight of more than about 200,000 and comprising hydrophobic protein molecules having a molecular weight of from 10,000 to 700,000 which have been cross-linked with a bifunctional or polyfunctional compound to produce said protein polymer, and having a plurality of molecules of a first member of a specific binding pair P1, and (ii) cross-linking said first polymer to a second polymer which comprises a plurality of molecules of a second member of a specific binding pair P2, and binding sites for a complex of an immune component and an analyte, said substance and said second polymer being cross-linked via binding of P1 and P2 to each other, and said first polymer being more hydrophobic than said second polymer.
(i) coating a substance on an insoluble carrier material, said substance containing a first polymer, said first polymer being a protein polymer having a molecular weight of more than about 200,000 and comprising hydrophobic protein molecules having a molecular weight of from 10,000 to 700,000 which have been cross-linked with a bifunctional or polyfunctional compound to produce said protein polymer, and having a plurality of molecules of a first member of a specific binding pair P1, and (ii) cross-linking said first polymer to a second polymer which comprises a plurality of molecules of a second member of a specific binding pair P2, and binding sites for a complex of an immune component and an analyte, said substance and said second polymer being cross-linked via binding of P1 and P2 to each other, and said first polymer being more hydrophobic than said second polymer.
2. A process of claim 1, wherein said protein polymer is a soluble protein more hydrophobic than both P1 and P2.
3. A process of claim 1, wherein said first polymer has a molecular weight of from 200,000 to 20,000,000.
4. A process of claim 1, 2 or 3, wherein said bifunctional compound is disuccinimidyl suberate.
5. A process of claim 1, 2 or 3, wherein said protein is bovine serum albumin or immune-gamma globulin.
6. A process of claim 4, wherein said protein is bovine serum albumin or immune-gamma globulin.
7. A process of claim 1, 2, 3 or 6, wherein said second polymer consists of cross-linked molecules of P2 and a homo-, heterobi- or polyfunctional linker.
8. A process of claim 5, wherein said second polymer consists of cross-linked molecules of P2 and a homo-, heterobi- or polyfunctional linker.
9. A process of claim 6, wherein said second polymer consists of cross-linked molecules of P2 and a homo-, heterobi- or polyfunctional linker.
10. A process of claim 1, 2 or 3, wherein said second polymer consists of molecules of P2.
11. A process of claim 1, 2 or 3, wherein said second polymer comprises a second component cross-linked with P2.
12. A process of claim 1, 2 or 3, wherein said second polymer comprises a hydrophobic protein.
13. A process of claim 1, 2 or 3, wherein said second polymer comprises a hydrophobic protein cross-linked to P2.
14. A process of claim 1, 2 or 3, wherein P1 and P2 are selected from the group consisting of biotin-avidin, biotin-streptavidin, antigen-antibody, hapten-antibody, protein-A-immune-.gamma.-globulin and protein G-immune-.gamma.-globulin.
15. A process of claim 1, 2, 3, 6, 8 or 9, wherein said insoluble solid carrier is glass, titanium dioxide, polystyrene, .gamma.-globulin activated polystyrene, paper or polyolefin.
16. A solid matrix useful in determining an analyte in a sample, comprising:
(i) an insoluble carrier material having coated thereon a first polymer having a molecular weight of more than about 200,000 comprising hydrophobic protein molecules having a molecular weight of from 10,000 to 700,000 which have been cross-linked with a bifunctional or polyfunctional compound to produce said first polymer and containing a plurality of molecules of a first member of a specific binding pair P1, and (ii) a second polymer comprising a plurality of molecules of a second member of a specific binding pair P2 cross-linked to said first polymer via bonds between P1 and P2, said first polymer being more hydrophobic than said second polymer and said second polymer further comprises binding sites for a complex containing said analyte.
(i) an insoluble carrier material having coated thereon a first polymer having a molecular weight of more than about 200,000 comprising hydrophobic protein molecules having a molecular weight of from 10,000 to 700,000 which have been cross-linked with a bifunctional or polyfunctional compound to produce said first polymer and containing a plurality of molecules of a first member of a specific binding pair P1, and (ii) a second polymer comprising a plurality of molecules of a second member of a specific binding pair P2 cross-linked to said first polymer via bonds between P1 and P2, said first polymer being more hydrophobic than said second polymer and said second polymer further comprises binding sites for a complex containing said analyte.
17. A solid phase matrix according to claim 16, wherein the first polymer is a conjugate of a soluble protein with a molecular weight of 200,000 to 20,000,000 and a plurality of biotin, avidin or streptavidin molecules.
18. A solid phase matrix according to claim 16, wherein the first polymer consists of biotin, avidin or streptavidin and a hydrophobed protein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3806431A DE3806431A1 (en) | 1988-02-29 | 1988-02-29 | METHOD FOR PRODUCING A SOLID PHASE MATRIX |
| DEP3806431.6 | 1988-02-29 |
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| Publication Number | Publication Date |
|---|---|
| CA1335265C true CA1335265C (en) | 1995-04-18 |
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ID=6348450
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000592220A Expired - Fee Related CA1335265C (en) | 1988-02-29 | 1989-02-27 | Solid phase matrices and a process for the production thereof |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0331127B1 (en) |
| JP (1) | JPH0830705B2 (en) |
| AT (1) | ATE90790T1 (en) |
| CA (1) | CA1335265C (en) |
| DE (2) | DE3806431A1 (en) |
| ES (1) | ES2058361T3 (en) |
| GR (1) | GR3009564T3 (en) |
| WO (1) | WO1989008259A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0488152A3 (en) * | 1990-11-30 | 1992-11-25 | Hitachi, Ltd. | Method for immunoassay and apparatus therefor |
| DE4041080A1 (en) | 1990-12-21 | 1992-06-25 | Behringwerke Ag | METHOD FOR DETERMINING AN ANALYT |
| JP2782695B2 (en) * | 1992-09-11 | 1998-08-06 | ダムビノヴァ，スヴェトラーナ・アレクサンドロフナ | Immunosorbent for diagnosis of neurosis and psychiatric disorders and its practical use |
| EP0699906B1 (en) | 1994-07-25 | 2002-04-24 | Roche Diagnostics GmbH | Method for detecting the contamination of a surface with an analyte |
| DE4434093A1 (en) * | 1994-09-23 | 1996-03-28 | Boehringer Mannheim Gmbh | Method for the qualitative and / or quantitative detection of a substance to be determined |
| DE19709090A1 (en) * | 1997-03-06 | 1998-09-10 | Boehringer Mannheim Gmbh | Analytical test element with liquid-filled blisters |
| DE19724787A1 (en) * | 1997-06-06 | 1998-12-10 | Biotez Berlin Buch Gmbh Bioche | Streptavidin / avidin coated surfaces |
| DE10020885A1 (en) * | 1999-05-03 | 2001-01-18 | Biotez Berlin Buch Gmbh | Preparation of a chemo- and thermo-stabile solid phase matrix for combining a polymerase chain reaction and a hybridization assay, by reacting a biotinylated cross-linking agent with a carrier material in strong basic or acid buffer |
| DE19927783A1 (en) | 1999-06-18 | 2000-12-21 | Roche Diagnostics Gmbh | Element for determining an analyte in a liquid, corresponding determination method using the element and kit for determining an analyte |
| US20230322996A1 (en) | 2021-11-08 | 2023-10-12 | Life Technologies As | Polymer-interaction molecule conjugates and methods of use |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4230685A (en) * | 1979-02-28 | 1980-10-28 | Northwestern University | Method of magnetic separation of cells and the like, and microspheres for use therein |
| US4282287A (en) * | 1980-01-24 | 1981-08-04 | Giese Roger W | Biochemical avidin-biotin multiple-layer system |
| US4478946A (en) * | 1981-07-02 | 1984-10-23 | South African Inventions Development Corporation | Carrier bound immunosorbent |
| FR2543972B1 (en) * | 1983-04-06 | 1985-12-27 | Immunotech Sa | METHOD FOR FIXING BIOLOGICAL MACROMOLECULES ON SUPPORTS |
| DE3446636A1 (en) * | 1984-12-20 | 1986-06-26 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR PRODUCING AN IMMUNREACTIVE POROUS CARRIER MATERIAL |
| WO1987004794A1 (en) * | 1986-02-06 | 1987-08-13 | Microbiological Associates, Inc. | Latex agglutination using avidin/biotin system |
| US4778751A (en) * | 1986-05-12 | 1988-10-18 | Diagnostic Products Corporation | Method for measuring antigens or antibodies in biological fluids using ligand labeled antigens or ligand labeled antibodies |
| DE3640412A1 (en) * | 1986-11-26 | 1988-06-09 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING A SPECIFICALLY BINDABLE SUBSTANCE |
-
1988
- 1988-02-29 DE DE3806431A patent/DE3806431A1/en not_active Withdrawn
-
1989
- 1989-02-27 CA CA000592220A patent/CA1335265C/en not_active Expired - Fee Related
- 1989-02-28 WO PCT/EP1989/000195 patent/WO1989008259A1/en unknown
- 1989-02-28 ES ES89103528T patent/ES2058361T3/en not_active Expired - Lifetime
- 1989-02-28 DE DE8989103528T patent/DE58904672D1/en not_active Expired - Lifetime
- 1989-02-28 AT AT89103528T patent/ATE90790T1/en not_active IP Right Cessation
- 1989-02-28 JP JP1502567A patent/JPH0830705B2/en not_active Expired - Lifetime
- 1989-02-28 EP EP89103528A patent/EP0331127B1/en not_active Expired - Lifetime
-
1993
- 1993-08-05 GR GR920403236T patent/GR3009564T3/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ES2058361T3 (en) | 1994-11-01 |
| WO1989008259A1 (en) | 1989-09-08 |
| DE58904672D1 (en) | 1993-07-22 |
| EP0331127B1 (en) | 1993-06-16 |
| JPH0830705B2 (en) | 1996-03-27 |
| EP0331127A1 (en) | 1989-09-06 |
| GR3009564T3 (en) | 1994-02-28 |
| DE3806431A1 (en) | 1989-09-07 |
| ATE90790T1 (en) | 1993-07-15 |
| JPH02500687A (en) | 1990-03-08 |
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