CA2005926C - Process for immobilising a protein on a solid phase - Google Patents
Process for immobilising a protein on a solid phaseInfo
- Publication number
- CA2005926C CA2005926C CA002005926A CA2005926A CA2005926C CA 2005926 C CA2005926 C CA 2005926C CA 002005926 A CA002005926 A CA 002005926A CA 2005926 A CA2005926 A CA 2005926A CA 2005926 C CA2005926 C CA 2005926C
- Authority
- CA
- Canada
- Prior art keywords
- solid phase
- aggregate
- protein
- homopolymer
- contacting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/813—Carrier is a saccharide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/813—Carrier is a saccharide
- Y10S530/814—Cellulose or derivatives thereof
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/815—Carrier is a synthetic polymer
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/815—Carrier is a synthetic polymer
- Y10S530/817—Entrapped within the carrier, e.g. gel, hollow fibre
Abstract
A process for immobilising a protein on a solid phase, comprises aggregating the protein to be immobilised, contacting it in a liquid with a hydrophilic solid phase and drying the solid phase, after contact has taken place; the solid phase thus prepared may be used for analytical determinations, for example, in diagnosis.
Description
X00 a9~~
The present invention is concerned with a process for immobilising proteins on a solid phase and with a protein-carrying solid phase so prepared, as well as with the use thereof, Proteins immobilised on solid phases are of great importance in various fields of technology. There may be mentioned, for example, the use of immobilised proteins in biotechnology, for example in bioreactors, and in biochemical production, for example for affinity chromatography.
Immobilised proteins have achieved a particular importance for analysis. Especially in the case of detection processes for the parameters relevant in medical diagnosis in body fluids, such as blood, plasma, serum, urine, saliva, liguor and the like, bioaffine reactions play an important part , i.e. those reactions in which a particular substance is specifically bound to a protein. Known substance/protein pairs which enter into such bioaffine reactions are, in general, for example antigen/antibody, carbohydrate/lectin, biotin/avidin and the liken If the protein is immobilised on a solid phase, corresponding substances which are specifically bindable with the protein and are present in a sample are bound thereon and are removed from the sample and determined qualitatively and/or quantitatively.
For example, for the detection of a partner of the antigen/antibody pair, there has been developed the principle of heterogenous immunoassay, many variants of ~~~~92~
The present invention is concerned with a process for immobilising proteins on a solid phase and with a protein-carrying solid phase so prepared, as well as with the use thereof, Proteins immobilised on solid phases are of great importance in various fields of technology. There may be mentioned, for example, the use of immobilised proteins in biotechnology, for example in bioreactors, and in biochemical production, for example for affinity chromatography.
Immobilised proteins have achieved a particular importance for analysis. Especially in the case of detection processes for the parameters relevant in medical diagnosis in body fluids, such as blood, plasma, serum, urine, saliva, liguor and the like, bioaffine reactions play an important part , i.e. those reactions in which a particular substance is specifically bound to a protein. Known substance/protein pairs which enter into such bioaffine reactions are, in general, for example antigen/antibody, carbohydrate/lectin, biotin/avidin and the liken If the protein is immobilised on a solid phase, corresponding substances which are specifically bindable with the protein and are present in a sample are bound thereon and are removed from the sample and determined qualitatively and/or quantitatively.
For example, for the detection of a partner of the antigen/antibody pair, there has been developed the principle of heterogenous immunoassay, many variants of ~~~~92~
which are known to the expert. It is common to all that a partner of the antigen/antibody pair is bound to a solid phase and with this immobilised partner is carried out a separation of at least a part of the corresponding binding partner from the remaining sample, The amount of the binding partner separated off or of the part of the binding partner not separated off but remaining in the sample is then determined on the solid phase or in the remaining sample, From the prior art, a series of processes is known for the immobilisation of proteins on solid phases. Thus, the fixing of a protein on a solid phase can take place by chemical or physical forces. P~Zethods for the pro-duction of covalent bindings between a solid carrier material and proteins to be bound thereon have been known for a long time, For example, European Patent Specification No, 0,274,911 describes the use of chemically reactive synthetic resin membranes which are able directly co-valently to bind proteins, However, this process for t he production of protein-carrying solid phases requires a long contact time between the membrane and the protein to be bound in order to allow the chemical reaction to proceed to completion. Furthermore, active positions not saturated with protein must, in a subsequent step, be occupied with an inert protein in order that no more free membrane active positions are present which could later negatively influence the use of the protein-laden solid phase.
r ~00~926 Known processes in which reactive groups of the solid phase are coupled with a bifunctional linker, to the remaining free function of which is then covalently bound the protein to be fixed, require still more time and still more process steps, However, with each process step, the risk also increases of the production of faulty batches and the production costs also increase, A problem of non-covalent fixing of proteins on a solid phase is the weaker binding. Thus, proteins which have been adsorbed from a solution on to a solid phase are relatively easily dissolved off from this. Suggest-ions for overcoming this problem are known. Federal Repz~.blic of Germany Patent Specification No. 34 46 636 describes, for example, processes for fixing an immune-reactive material on a porous carrier material. For the avoidance of adhesion problems on the carrier material, the fixing is thereby achieved by allowing an immune reaction to take place between the tyro partners of an immune reaction, i,e, between an antibody and an antigen or hapten. An immune complex mesh is thus formed which contains the protein to be bound (antibody or antigen) and binds on to the solid phase, The disadvantage of this fixing process is that, apart from t he protein to be bound, a further expensive-material (antibody or antigen) is also needed, ~~urthermore, the preparation of the batch must be carried out exactly in order to achieve an optimum binding on the solid phase, 200~92~
In British Patent Specification No. 1,505,400, it is suggested to cross-link an immunologically active protein and then to adsorb it on polystyrene latex particles, the adsorption being carried out in the latex emulsion. After the binding of a part of the protein on t he latex particles, these are centrifuged off and washed several times. The protein-carrying polystyrene particles are stored as a suspension in buffered aqueous solutions and used for separation reactions) European Patent Specification No. 0,122,209 describes a process for binding biological macromolecules on to solid phases which comprises polymerising the macro-molecules to be fixed, incubating for several hours together ~,rith hydrophobic carrier materials, for example polystyrene, and, after binding of a part of the polymer-ised macromolecules on to the carrier material, washing this several times befoxe use or storage.
The two above-mentioned processes do not result in a satisfactory adhesion of proteins, a specially of specifically bindable, i.e, bioaffine substances to the carrier. Furthermore, time-consuming and laborious incubation and washing steps are necessary for fixing the protein on to the carrier. European Patent Specif-ication N o. 0,269,092 is concerned with a process for improving the adhesion in comparison with t he two above-mentioned processes. For this purpose, the protein to be fixed is fixed covalently to a hydrophobic carrier protein and the complex obtained is adsorbed on a r hydrophobic solid phase. By utilisation of the hydro-phobic exchange action between the solid phase and t he carrier protein, an especially advantageous fixing is thereby achieved, It is common to the three last-mentioned publications that hydrophobic carrier materials are exclusively used for ttie non-covalent fixing of proteins which consider-ably limits the choice of carrier materials, Especially for the use of protein-carrying solid phases in aqueous liquids, such as are all biological fluids, hydrophilic materials are often to be preferred because of their better wettability.
Starting from this prior art, the present invention seeks to bind proteins on the carrier materials which are substantially insoluble in water by means of a process which, even on a technical scale, is simple and quick to carry out in order to obtain in this way protein-carrying solid phases which offer wide possibil-ities for use in the binding and possible separation of specifically bindable substances from liquid samples, Thus, according to the present invention, there is provided a process for the immobilisation of a protein on a solid phase, wherein the protein to be immobilised is aUgregated, contacted in a liquid with a hydrophilic solid phase and the solid phase, after contact has taken place, is dried.
By means of the process according to the present inventions in principle all proteins can be immobilised 200~9~6 which can be aggregated chemically or physically to higher molecular weights. By chemical aggregation is thereby to be understood every molecular weight increase which is achieved by means of chemical agents. Thus, proteins can,for example, be homopolymerised, for example by the addition of carbodiimides. However, they cans for example, also be joined or cross-linked with one another by means of polyfunctional molecules, so-called linkers. The palette of chemical linkers opens up the possibility of a greater variability of ,the aggregates, for example with regard to the accessibility of particular protein positions, so-called epitopes, to substances which are specifically bindable with t he protein or with regard to the adhesion ability of the protein aggregate to the solid phase. The method of cross-linking proteins by means of linkers is well known from t he prior art. For example, British Patent Specification Tto. 1,505,400 and European Patent Specifications rlos. 0,122,209 and 0,269,092 describe such processes. For the process according to the present invention, suitable linkers which have provided to be useful include disuccinidyl suberate, S-acetylmercapto-~zccinic acid anhydride and maleinimidohexanoyl hydroxy-succinimide. Disuccinidyl suberate is especially preferred according to the present invention for the aggregation of human albumin.
By physical aggregation is to be understood every molecular weight increase which is achieved without the use of chemical agents. For example, it is known thermally ~0050~0 to aggregate proteins, for example albumin, and thus to increase the molecular weight thereof (see European Patent Specification No. 0,269,092). Thermally aggregated albumin is, in the meaning of the present invention, an especially well immobilisable protein.
When the prepared protein aggregate is to be lyo-philised before its application to solid phase, for example for storage, it is recommended possibly to add a stabiliser to this before lyophilisation, as is often the case in the lyophilisation of protein solutions, which increases the storage stability of the lyophilised protein aggregate and its solubility in the case of reconstitution with water or buffer. the nature and amount of these substances are dependent upon the nature of the particular protein. In general, these materials possibly added to t he protein aggregate solution should not have a negative influence on the immobilisation process and especially onthe bindability of the protein aggregate on to the solid phase. Appropriate materials include, for example, saccharose, trehalose, mannose, dextrans and similar carbohydrates, as well as proteins, for example crotein C~ collagen and bovine serum albumin.
Saccharose has proved to be particularly advantageous, especially in the case of human albumin cross-linked with disuccinidyl suberate. A typical concentration range for the stabilisers possibly to be added to the protein aggregate solution to be lyophilised and/or of the solubilising agents is 2 to 20~ by weight. For the ~~0;~9~~
above-mentioned case of the addition of saccharose to a solution of human albumin cross-linked with disuccinidyl suberate, the preferred concentration is from 4 to 10~; by weight. In the case of the addition of stabilisers and/or of solubilising agents to the protein aggregate solution to be lyophilised, care is to be taken that, in the case of the use of the reconstituted lyophilisate for loading a solid phase in the meaning of the present invention, the concentration of stabiliser and/or solubiliser should not be so high that the bindability of the protein aggregate on to the solid phase is negatively influenced.
For example, in the case of the immobilisation of human albumin cross-linked with disuccinidyl suberate, the concentration of saccharose in the protein aggregate solution should be less than 2g~ by weight, As proteins, according to the process of the present invention, there can be immobilised not only naturally-occurring proteins and proteins isolated from natural sources but also synthetically prepared proteins.
Examples of proteins which can be especially yell immobilised according to the present invention include albumin, immunoglobulins, transferrin and collagen.
Accordin~ to the present invention, those proteins can also be fixed on to a solid phase which proteins contain a bound non-protein part, for example biotin or a carbohydrate, In this way, according to the present invention, non-proteins can also be immobilised on solid phases when they have previously been bound to a protein which can act as an anchor on the solid phase for the non-protein ~oo~o~o part. In the same way, proteins can naturally also be bound to a protein acting other than as an anchor to the solid phase, The strength of the immobilisation of the protein on the solid phase according to the present invention is dependent upon various factors: on the one hand upon t he particular protein to be fixed and on the other hand upon the nature of the carrier material. With regard to the protein, it has been found that the adhesion to the solid phase increases with increasing molecular weight. No general minimum size can be Uiven for the achievement of a particular binding strength. It depends upon the nature of the particular protein to be bound and can easily be determined. Thus, for example, in the case of albumin, an aggregate of 4 to 5 albumin units already shows a good immobilisability in t he meaninn of the present invention and an aggregate of at least l0 albumin units is especially advantageous, Preferred solid phases in the meaninn of the present invention are all those materials which are su~stant-ially insoluble in water and which are more hydrophilic than the aggregated protein to be immobilised Appropriate materials include, for example, polyesters, sulphite cellulose, regenerated cellulose, linters, nitrocellulose, cellulose acetate and solid phases based on nylon. They can be present in any desired form, for example powders, grains fibres, fleece or films, Fleece based on cellulose is especially preferred as solid phase iri the meaning - 2(~~~9~6 o-of the present invention.
According to the process of the present invention, the aggregated protein is contacted in a liquid with the solid phase. The aggregated protein is thereby present in dissolved form, preferably in an agueous liquid, for example a buffer solution. If fleecesare used as solid phases, it has proved to be especially advantageous to impregnate these with a solution of the aggregated protein in that they are dipped into the solution and saturated therein with the solution.
As buffer solutions, there can thereby be used all those which are knocun not to damage the protein in question or its biological activity. For example, for human albumin, phosphate or I~PES buffer with a pH value of from about 6 to about 9 and preferably of from about 7 to about 8 have proved to be especially useful as solvent For the achievement of a food adhesion of the aggregated protein to the solid phase, it is important that, after contacting the protein aggregate with the solid phase, the solid phase carrying the protein is dried. By drying is thereby to be understood all special measures for removing liquid as far as possible. As dry in the meaning of the present invention is designated, in general, a hydrophilic solid phase with a residual moisture content of less than '7°,~ by weight at 20°C, and 50~ relative humidity. The residual moisture content below which "dry" is to be understood is, in the ~oo~o~s particular case, dependent upon the chosen solid phase, Thus, for example, cellulose fleece in the case of a residual moisture content of less than 7~ by weight and a mixed fleece of cellulose and polyester fibres in the weight ratio of 50:50 of less than 5~ by weight are to be called dry. In order to achieve such a degree of drying in the case of hydrophilic solid phases in an economically acceptable drying time, as a rule heat must be applie d Thus, according to the process of the present invention, the drying of protein-carrying fleece is advantageously carried out at a temperature of from 30 to 80°C, If the dryintime is not decisive for the choice of the process, drying can also be carried out at ambient temperature in, for example, a current of air, surprisingly, it has been ascertained that the ac.hieve-ment of such a low residual moisture content of the solid phase carryinthe protein is decisive for the excellent adhesion of the protein on the carrier. Hiher residual moisture contents lead to a weaker fixing of the protein on the solid phase, The special advanta;e of the process according to the present invention lies in its simplicity. It suffices to dip the hydrophilic carrier material, especially 'hydro-philic fleece, in a solution of the aggregated protein to be fixed in order to saturate it with liquid. After impregnation has taken place, the carrier material is removed from the protein solution and dried, After drying, the protein agregate is firmly bound to the solid phase, In this way, it is possible to achieve 20U;~~2~
a homogeneous loading density of the solid phase with protein, the amount of immobilised protein thereby being known exactly.
The amount of protein which can be so bound to a solid phase is substantially higher than can be explained by adsorption. Thus, when after carrying out of the process according to the present invention, after the drying no protein can be eluted from the solid phase by an additional washing step, whereas without drying, i.e. when a simple adsorption of the protein aggregate on the solid phase was carried out, the greater part of the adsorbed protein is again eluted by washing. Therefore, according to the process of the present invention, a washinstep is unnecessar9 vahen the concentration of the protein so~.ution is so chosen that the total protein is immobilised after the drying. This protein concentration is dependent upon the nature of the particular carrier material. The limiting concentration which is just completely immobil-ised can easily be determined by simple experiments.
A further advantage of the process accordinto the present inven'~ion is that the composition of the impreg-nation solution can be freely selected in wide limits without disadvantageously influencing the bindinprop-erties of the protein to be immobilised. Thus, for example, the nature and concentration of the buffer, the pH value, ionic strength and possibly stabilisers and/or solubilisers to be added can preponderantly be selected solely according to the properties of the particular dissolved protein ~oo~s~s -'13 -aggregate. This choice usually has no influence cn the strength of the binding of the immobilised protein aggregate.
Solid phases produced by the process according to the present invention are very well suited for binding sub-stances which are specifically bindable with the immobil-i wed protein and possibly for the removal thereof from liquids. The most varied fields of use are conceivable, for example in biochemical production as carriers of immobilised proteins in bioreactors or for affinity chromatography for the enrichment and/or separation of specifically bindable substances, Protein-carrying solid phases produced according to the present invention are especially preferably used for analytical determinations of component materials in liquid samples, They are quite especially preferred for bioaffine determinations of component materials in body fluids, for example blood, plasma, serum, urine, liquor, saliva and the like, as were described hereinbefore. For hetero-genous immunoassays, the protein-carrying solid phases according to the present invention are especially well suited, particularly for those which, in all, are carried out carrier-bound, for example on a test strip) ...
Thus in accordance with a particular embodiment of the invention there is provided a process for immobilizing a protein containing substance onto a solid phase comprising: (i) forming a water soluble aggregate of a polymer containing more than one molecule of a particular protein; (ii) contacting a liquid containing said aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said aggregate immobilized and non-covalently bound thereon.
In another aspect of the invention there is provided a solid phase containing non-covalently bound protein.
In accordance with one embodiment of this aspect there is provided a solid phase having a composite comprising protein aggregates bound thereto, comprising: a hydrophilic solid phase having non-covalently bound thereto a protein aggregate, wherein said aggregate is formed by aggregating more than one molecule of a particular protein to form a polymer containing aggregate prior to contacting said aggregate with said solid phase, wherein said solid phase is dried following contact of said solid phase with said polymer containing aggregate.
In accordance with another embodiment of this aspect there is provided a solid phase having a composite comprising protein aggregates bound thereto, comprising: a hydrophilic solid phase having non-covalently bound thereto a protein homopolymer aggregate, wherein said homopolymer aggregate is formed prior to contacting said homopolymer aggregate to said solid phase, wherein said solid phase is dried following contact of said solid phase with said homopolymer aggregate.
_.
In accordance with still another embodiment of this aspect there is provided a solid phase having a composite protein aggregates bound thereto comprising a hydrophilic solid phase having non-covalently bound thereto a protein aggregate, wherein said aggregate is formed by mixing more than one molecule of a particular protein with a polyfunctional linker to form a protein containing aggregate prior to contacting said protein aggregate with said hydrophilic solid phase, wherein said solid phase is dried following contact of said solid phase with said protein aggregate.
In another aspect of the invention there is provided a process for immobilizing a protein containing substance onto a solid phase comprising:
(i) forming a water soluble protein homopolymer aggregate; (ii) contacting a liquid containing said protein homopolymer aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said protein homopolymer aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said protein homopolymer aggregate immobilized and non-covalently bound thereon.
In still another aspect of the invention there is provided a method for determining an analyte in a liquid sample, comprising contacting the liquid sample with a solid phase of the invention, wherein the aggregate comprises a substance which binds with the analyte and determining the analyte bound to the aggregate.
A
The following Examples are given for the purpose of illustrating the present invention. Reference is also made to the accompanying drawings in which Figures 1 and 2 are standard curved obtained with the use of the solid phases according to the present invention:
r, '-Example l, Cross-linking of human serum albumin (HSA) by means of disuccinidyl suberate (DSS) to give poly-human serum albumin (pHSA).
1.5 g. HSA is placed in 30 ml, potassium phosphate buffer (200 mrl~ pH 8.0) and mixed within 2 hours with 2,5 ml, of a solution of 50 mg, DSS/ml. dioxan.,aAfter completion of the cross-linking reaction, dialysis is carried out against a 50C fold volume of potassium phosphate buffer (20 mNI, pH ~,2). The high molecular weight fraction (pHSA) with a molecular weight of more than 650,000 Dalton is separated on aup.erose 6 R (Pharmacia, Freiburg, Federal Republic of Germany) by gel filtration ands after the addition of 6 mg. saccharose/mg. of protein, is lyophilised, Example 2, Immobilisation according to the present invention of human serum albumin and comparison of the strength of adhesion of the so immobilised protein with adsorbed rotein.
6 x 6 mm2 sized and 0,5 mm, thick pieces of fleece of 50;o polyester/SCg; linters are impregnated with 15 ~.1, of a solution of 250 mg./litre pHSA from F,xample 1 in ZO mM
sodium phosphate buffer (pH 7.5) and a) dried for 30 minutes at 50o C and washed three times with, in each case, 25 /,~.1, sodium phosphate buffer (ZO mM, pH 7.5) in a centrifuge and subsequently centrifuged out or A
__ - 18 -b) after 10 minutes, centrifuged out in a centrifuge and washed twice with, in each case, 25 r..l. sodium phosphate buffer (10 mrl, pH ?.5).
The wash centrifugates from a) and b) are, in each case, combined and, in each case, mixed-with 300 mU anti-HSA-IgG-~3-galactosidase conjugate (160 ~.1.) and shaken for l0 minuteso Then, in each case, 200 ~,1, are trans-ferred into microtitre plate cups, the walls of cahich are loaded with HSA. After incubation for 1 hour, the cups are washed with phosphate-buffered physiological sodium chloride solution, developed with o-nitrophenylgalactoside and, after l0 minutes, the extinction is measured at 405 nm~
The quantitative determination of the pHSA eluted from the fleece takes place by means of a calibration curve made at the same time.
The amount of pHSA with cahich each piece of fleece was contacted was 3.~5 ~.g. After the elution, the following amounts were determined in the combined wash centri-fu;ates:
process pHSA in the eluate referred to the eluate impregnation solution a) 0.012 duo. 0.3~' b) 2.64 ~ug~ ?l~;
The protein immobilised according to the present invention is, because of the drying step, substantially more firmly bound to the solid phase than the solely adsorbed protein which, to the greater part, can again be washed out from the solid phase.
Example 3, a) Preparation and cross-linking of rabbit I~G-T3 conjugate.
r ~00~926 Known processes in which reactive groups of the solid phase are coupled with a bifunctional linker, to the remaining free function of which is then covalently bound the protein to be fixed, require still more time and still more process steps, However, with each process step, the risk also increases of the production of faulty batches and the production costs also increase, A problem of non-covalent fixing of proteins on a solid phase is the weaker binding. Thus, proteins which have been adsorbed from a solution on to a solid phase are relatively easily dissolved off from this. Suggest-ions for overcoming this problem are known. Federal Repz~.blic of Germany Patent Specification No. 34 46 636 describes, for example, processes for fixing an immune-reactive material on a porous carrier material. For the avoidance of adhesion problems on the carrier material, the fixing is thereby achieved by allowing an immune reaction to take place between the tyro partners of an immune reaction, i,e, between an antibody and an antigen or hapten. An immune complex mesh is thus formed which contains the protein to be bound (antibody or antigen) and binds on to the solid phase, The disadvantage of this fixing process is that, apart from t he protein to be bound, a further expensive-material (antibody or antigen) is also needed, ~~urthermore, the preparation of the batch must be carried out exactly in order to achieve an optimum binding on the solid phase, 200~92~
In British Patent Specification No. 1,505,400, it is suggested to cross-link an immunologically active protein and then to adsorb it on polystyrene latex particles, the adsorption being carried out in the latex emulsion. After the binding of a part of the protein on t he latex particles, these are centrifuged off and washed several times. The protein-carrying polystyrene particles are stored as a suspension in buffered aqueous solutions and used for separation reactions) European Patent Specification No. 0,122,209 describes a process for binding biological macromolecules on to solid phases which comprises polymerising the macro-molecules to be fixed, incubating for several hours together ~,rith hydrophobic carrier materials, for example polystyrene, and, after binding of a part of the polymer-ised macromolecules on to the carrier material, washing this several times befoxe use or storage.
The two above-mentioned processes do not result in a satisfactory adhesion of proteins, a specially of specifically bindable, i.e, bioaffine substances to the carrier. Furthermore, time-consuming and laborious incubation and washing steps are necessary for fixing the protein on to the carrier. European Patent Specif-ication N o. 0,269,092 is concerned with a process for improving the adhesion in comparison with t he two above-mentioned processes. For this purpose, the protein to be fixed is fixed covalently to a hydrophobic carrier protein and the complex obtained is adsorbed on a r hydrophobic solid phase. By utilisation of the hydro-phobic exchange action between the solid phase and t he carrier protein, an especially advantageous fixing is thereby achieved, It is common to the three last-mentioned publications that hydrophobic carrier materials are exclusively used for ttie non-covalent fixing of proteins which consider-ably limits the choice of carrier materials, Especially for the use of protein-carrying solid phases in aqueous liquids, such as are all biological fluids, hydrophilic materials are often to be preferred because of their better wettability.
Starting from this prior art, the present invention seeks to bind proteins on the carrier materials which are substantially insoluble in water by means of a process which, even on a technical scale, is simple and quick to carry out in order to obtain in this way protein-carrying solid phases which offer wide possibil-ities for use in the binding and possible separation of specifically bindable substances from liquid samples, Thus, according to the present invention, there is provided a process for the immobilisation of a protein on a solid phase, wherein the protein to be immobilised is aUgregated, contacted in a liquid with a hydrophilic solid phase and the solid phase, after contact has taken place, is dried.
By means of the process according to the present inventions in principle all proteins can be immobilised 200~9~6 which can be aggregated chemically or physically to higher molecular weights. By chemical aggregation is thereby to be understood every molecular weight increase which is achieved by means of chemical agents. Thus, proteins can,for example, be homopolymerised, for example by the addition of carbodiimides. However, they cans for example, also be joined or cross-linked with one another by means of polyfunctional molecules, so-called linkers. The palette of chemical linkers opens up the possibility of a greater variability of ,the aggregates, for example with regard to the accessibility of particular protein positions, so-called epitopes, to substances which are specifically bindable with t he protein or with regard to the adhesion ability of the protein aggregate to the solid phase. The method of cross-linking proteins by means of linkers is well known from t he prior art. For example, British Patent Specification Tto. 1,505,400 and European Patent Specifications rlos. 0,122,209 and 0,269,092 describe such processes. For the process according to the present invention, suitable linkers which have provided to be useful include disuccinidyl suberate, S-acetylmercapto-~zccinic acid anhydride and maleinimidohexanoyl hydroxy-succinimide. Disuccinidyl suberate is especially preferred according to the present invention for the aggregation of human albumin.
By physical aggregation is to be understood every molecular weight increase which is achieved without the use of chemical agents. For example, it is known thermally ~0050~0 to aggregate proteins, for example albumin, and thus to increase the molecular weight thereof (see European Patent Specification No. 0,269,092). Thermally aggregated albumin is, in the meaning of the present invention, an especially well immobilisable protein.
When the prepared protein aggregate is to be lyo-philised before its application to solid phase, for example for storage, it is recommended possibly to add a stabiliser to this before lyophilisation, as is often the case in the lyophilisation of protein solutions, which increases the storage stability of the lyophilised protein aggregate and its solubility in the case of reconstitution with water or buffer. the nature and amount of these substances are dependent upon the nature of the particular protein. In general, these materials possibly added to t he protein aggregate solution should not have a negative influence on the immobilisation process and especially onthe bindability of the protein aggregate on to the solid phase. Appropriate materials include, for example, saccharose, trehalose, mannose, dextrans and similar carbohydrates, as well as proteins, for example crotein C~ collagen and bovine serum albumin.
Saccharose has proved to be particularly advantageous, especially in the case of human albumin cross-linked with disuccinidyl suberate. A typical concentration range for the stabilisers possibly to be added to the protein aggregate solution to be lyophilised and/or of the solubilising agents is 2 to 20~ by weight. For the ~~0;~9~~
above-mentioned case of the addition of saccharose to a solution of human albumin cross-linked with disuccinidyl suberate, the preferred concentration is from 4 to 10~; by weight. In the case of the addition of stabilisers and/or of solubilising agents to the protein aggregate solution to be lyophilised, care is to be taken that, in the case of the use of the reconstituted lyophilisate for loading a solid phase in the meaning of the present invention, the concentration of stabiliser and/or solubiliser should not be so high that the bindability of the protein aggregate on to the solid phase is negatively influenced.
For example, in the case of the immobilisation of human albumin cross-linked with disuccinidyl suberate, the concentration of saccharose in the protein aggregate solution should be less than 2g~ by weight, As proteins, according to the process of the present invention, there can be immobilised not only naturally-occurring proteins and proteins isolated from natural sources but also synthetically prepared proteins.
Examples of proteins which can be especially yell immobilised according to the present invention include albumin, immunoglobulins, transferrin and collagen.
Accordin~ to the present invention, those proteins can also be fixed on to a solid phase which proteins contain a bound non-protein part, for example biotin or a carbohydrate, In this way, according to the present invention, non-proteins can also be immobilised on solid phases when they have previously been bound to a protein which can act as an anchor on the solid phase for the non-protein ~oo~o~o part. In the same way, proteins can naturally also be bound to a protein acting other than as an anchor to the solid phase, The strength of the immobilisation of the protein on the solid phase according to the present invention is dependent upon various factors: on the one hand upon t he particular protein to be fixed and on the other hand upon the nature of the carrier material. With regard to the protein, it has been found that the adhesion to the solid phase increases with increasing molecular weight. No general minimum size can be Uiven for the achievement of a particular binding strength. It depends upon the nature of the particular protein to be bound and can easily be determined. Thus, for example, in the case of albumin, an aggregate of 4 to 5 albumin units already shows a good immobilisability in t he meaninn of the present invention and an aggregate of at least l0 albumin units is especially advantageous, Preferred solid phases in the meaninn of the present invention are all those materials which are su~stant-ially insoluble in water and which are more hydrophilic than the aggregated protein to be immobilised Appropriate materials include, for example, polyesters, sulphite cellulose, regenerated cellulose, linters, nitrocellulose, cellulose acetate and solid phases based on nylon. They can be present in any desired form, for example powders, grains fibres, fleece or films, Fleece based on cellulose is especially preferred as solid phase iri the meaning - 2(~~~9~6 o-of the present invention.
According to the process of the present invention, the aggregated protein is contacted in a liquid with the solid phase. The aggregated protein is thereby present in dissolved form, preferably in an agueous liquid, for example a buffer solution. If fleecesare used as solid phases, it has proved to be especially advantageous to impregnate these with a solution of the aggregated protein in that they are dipped into the solution and saturated therein with the solution.
As buffer solutions, there can thereby be used all those which are knocun not to damage the protein in question or its biological activity. For example, for human albumin, phosphate or I~PES buffer with a pH value of from about 6 to about 9 and preferably of from about 7 to about 8 have proved to be especially useful as solvent For the achievement of a food adhesion of the aggregated protein to the solid phase, it is important that, after contacting the protein aggregate with the solid phase, the solid phase carrying the protein is dried. By drying is thereby to be understood all special measures for removing liquid as far as possible. As dry in the meaning of the present invention is designated, in general, a hydrophilic solid phase with a residual moisture content of less than '7°,~ by weight at 20°C, and 50~ relative humidity. The residual moisture content below which "dry" is to be understood is, in the ~oo~o~s particular case, dependent upon the chosen solid phase, Thus, for example, cellulose fleece in the case of a residual moisture content of less than 7~ by weight and a mixed fleece of cellulose and polyester fibres in the weight ratio of 50:50 of less than 5~ by weight are to be called dry. In order to achieve such a degree of drying in the case of hydrophilic solid phases in an economically acceptable drying time, as a rule heat must be applie d Thus, according to the process of the present invention, the drying of protein-carrying fleece is advantageously carried out at a temperature of from 30 to 80°C, If the dryintime is not decisive for the choice of the process, drying can also be carried out at ambient temperature in, for example, a current of air, surprisingly, it has been ascertained that the ac.hieve-ment of such a low residual moisture content of the solid phase carryinthe protein is decisive for the excellent adhesion of the protein on the carrier. Hiher residual moisture contents lead to a weaker fixing of the protein on the solid phase, The special advanta;e of the process according to the present invention lies in its simplicity. It suffices to dip the hydrophilic carrier material, especially 'hydro-philic fleece, in a solution of the aggregated protein to be fixed in order to saturate it with liquid. After impregnation has taken place, the carrier material is removed from the protein solution and dried, After drying, the protein agregate is firmly bound to the solid phase, In this way, it is possible to achieve 20U;~~2~
a homogeneous loading density of the solid phase with protein, the amount of immobilised protein thereby being known exactly.
The amount of protein which can be so bound to a solid phase is substantially higher than can be explained by adsorption. Thus, when after carrying out of the process according to the present invention, after the drying no protein can be eluted from the solid phase by an additional washing step, whereas without drying, i.e. when a simple adsorption of the protein aggregate on the solid phase was carried out, the greater part of the adsorbed protein is again eluted by washing. Therefore, according to the process of the present invention, a washinstep is unnecessar9 vahen the concentration of the protein so~.ution is so chosen that the total protein is immobilised after the drying. This protein concentration is dependent upon the nature of the particular carrier material. The limiting concentration which is just completely immobil-ised can easily be determined by simple experiments.
A further advantage of the process accordinto the present inven'~ion is that the composition of the impreg-nation solution can be freely selected in wide limits without disadvantageously influencing the bindinprop-erties of the protein to be immobilised. Thus, for example, the nature and concentration of the buffer, the pH value, ionic strength and possibly stabilisers and/or solubilisers to be added can preponderantly be selected solely according to the properties of the particular dissolved protein ~oo~s~s -'13 -aggregate. This choice usually has no influence cn the strength of the binding of the immobilised protein aggregate.
Solid phases produced by the process according to the present invention are very well suited for binding sub-stances which are specifically bindable with the immobil-i wed protein and possibly for the removal thereof from liquids. The most varied fields of use are conceivable, for example in biochemical production as carriers of immobilised proteins in bioreactors or for affinity chromatography for the enrichment and/or separation of specifically bindable substances, Protein-carrying solid phases produced according to the present invention are especially preferably used for analytical determinations of component materials in liquid samples, They are quite especially preferred for bioaffine determinations of component materials in body fluids, for example blood, plasma, serum, urine, liquor, saliva and the like, as were described hereinbefore. For hetero-genous immunoassays, the protein-carrying solid phases according to the present invention are especially well suited, particularly for those which, in all, are carried out carrier-bound, for example on a test strip) ...
Thus in accordance with a particular embodiment of the invention there is provided a process for immobilizing a protein containing substance onto a solid phase comprising: (i) forming a water soluble aggregate of a polymer containing more than one molecule of a particular protein; (ii) contacting a liquid containing said aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said aggregate immobilized and non-covalently bound thereon.
In another aspect of the invention there is provided a solid phase containing non-covalently bound protein.
In accordance with one embodiment of this aspect there is provided a solid phase having a composite comprising protein aggregates bound thereto, comprising: a hydrophilic solid phase having non-covalently bound thereto a protein aggregate, wherein said aggregate is formed by aggregating more than one molecule of a particular protein to form a polymer containing aggregate prior to contacting said aggregate with said solid phase, wherein said solid phase is dried following contact of said solid phase with said polymer containing aggregate.
In accordance with another embodiment of this aspect there is provided a solid phase having a composite comprising protein aggregates bound thereto, comprising: a hydrophilic solid phase having non-covalently bound thereto a protein homopolymer aggregate, wherein said homopolymer aggregate is formed prior to contacting said homopolymer aggregate to said solid phase, wherein said solid phase is dried following contact of said solid phase with said homopolymer aggregate.
_.
In accordance with still another embodiment of this aspect there is provided a solid phase having a composite protein aggregates bound thereto comprising a hydrophilic solid phase having non-covalently bound thereto a protein aggregate, wherein said aggregate is formed by mixing more than one molecule of a particular protein with a polyfunctional linker to form a protein containing aggregate prior to contacting said protein aggregate with said hydrophilic solid phase, wherein said solid phase is dried following contact of said solid phase with said protein aggregate.
In another aspect of the invention there is provided a process for immobilizing a protein containing substance onto a solid phase comprising:
(i) forming a water soluble protein homopolymer aggregate; (ii) contacting a liquid containing said protein homopolymer aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said protein homopolymer aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said protein homopolymer aggregate immobilized and non-covalently bound thereon.
In still another aspect of the invention there is provided a method for determining an analyte in a liquid sample, comprising contacting the liquid sample with a solid phase of the invention, wherein the aggregate comprises a substance which binds with the analyte and determining the analyte bound to the aggregate.
A
The following Examples are given for the purpose of illustrating the present invention. Reference is also made to the accompanying drawings in which Figures 1 and 2 are standard curved obtained with the use of the solid phases according to the present invention:
r, '-Example l, Cross-linking of human serum albumin (HSA) by means of disuccinidyl suberate (DSS) to give poly-human serum albumin (pHSA).
1.5 g. HSA is placed in 30 ml, potassium phosphate buffer (200 mrl~ pH 8.0) and mixed within 2 hours with 2,5 ml, of a solution of 50 mg, DSS/ml. dioxan.,aAfter completion of the cross-linking reaction, dialysis is carried out against a 50C fold volume of potassium phosphate buffer (20 mNI, pH ~,2). The high molecular weight fraction (pHSA) with a molecular weight of more than 650,000 Dalton is separated on aup.erose 6 R (Pharmacia, Freiburg, Federal Republic of Germany) by gel filtration ands after the addition of 6 mg. saccharose/mg. of protein, is lyophilised, Example 2, Immobilisation according to the present invention of human serum albumin and comparison of the strength of adhesion of the so immobilised protein with adsorbed rotein.
6 x 6 mm2 sized and 0,5 mm, thick pieces of fleece of 50;o polyester/SCg; linters are impregnated with 15 ~.1, of a solution of 250 mg./litre pHSA from F,xample 1 in ZO mM
sodium phosphate buffer (pH 7.5) and a) dried for 30 minutes at 50o C and washed three times with, in each case, 25 /,~.1, sodium phosphate buffer (ZO mM, pH 7.5) in a centrifuge and subsequently centrifuged out or A
__ - 18 -b) after 10 minutes, centrifuged out in a centrifuge and washed twice with, in each case, 25 r..l. sodium phosphate buffer (10 mrl, pH ?.5).
The wash centrifugates from a) and b) are, in each case, combined and, in each case, mixed-with 300 mU anti-HSA-IgG-~3-galactosidase conjugate (160 ~.1.) and shaken for l0 minuteso Then, in each case, 200 ~,1, are trans-ferred into microtitre plate cups, the walls of cahich are loaded with HSA. After incubation for 1 hour, the cups are washed with phosphate-buffered physiological sodium chloride solution, developed with o-nitrophenylgalactoside and, after l0 minutes, the extinction is measured at 405 nm~
The quantitative determination of the pHSA eluted from the fleece takes place by means of a calibration curve made at the same time.
The amount of pHSA with cahich each piece of fleece was contacted was 3.~5 ~.g. After the elution, the following amounts were determined in the combined wash centri-fu;ates:
process pHSA in the eluate referred to the eluate impregnation solution a) 0.012 duo. 0.3~' b) 2.64 ~ug~ ?l~;
The protein immobilised according to the present invention is, because of the drying step, substantially more firmly bound to the solid phase than the solely adsorbed protein which, to the greater part, can again be washed out from the solid phase.
Example 3, a) Preparation and cross-linking of rabbit I~G-T3 conjugate.
3 g. Rabbit IgG are dissolved in 30C mlo potassium phosphate buffer (100 mM, pH 8.5) and mixed with a solution of 58 mg. N-tert.-butyloxycarbonyltriiodo-thyronine N'-hydroxysuccinimide ester (BOC-T3-N'-hydrOxysuccinimide ester) in 30 ml, dioxan. After a reaction time of 2 hours, the protein solution is dialysed against a 200 fold volume of 20 mM potassium phosphate buffer (pH ~.8) and adjusted via ultra-filtration (Amicon YhI l00 '~ membrane) to a concentration of 50 mg./ml.
For the cross-linkin~, to the batch are slo~-~ly added 35 ml, disuccinidyl suberate (concentration ZO mg, di-succinidyl suberate/ml. dioxan). After completion of the cross-linking reaction, dialysis is carried out against a 500 fold volume of potassium phosphate buffer (50 mM, pH 7.2) and the fraction eluted on Superose 6 '~ in the exclusion volume separated off, stabilised with saccharose (concentration 6 mg. saccharose/mg. IgG) and lyophilised.
b) Triiodothyronine (T3) test.
A fleece of 605 sulphite cellulose and 40~' linters is impregnated with cross-linked rabbit IgG-T3 conjugate from Example 3a) in a concentration of 250 ~.o./ml. in A
50 mP~I potassium phosphate buffer (pH 7.2) and, after complete saturation, dried at 50oC, for 60 minutes in a circulating air cabinet. In each case, 50 f.,~.l~ of sheep anti-T3-IgG-(3-galactosidase conjugate (prepared analogously to the process described in J. Immunoassay, 4, 209-32'7/
2983)(120 mU in phosphate-buffered physiological sodium chloride solution, 5 g./litre bovine serum albumin) are incubated for 5,minutes with 50 ~,1, of a T3 standard series and subsequently pipetted on to the above-produced pieces of fleece (8 x 8 mm., thickness 0.5 mm,) of the T3 matrix, After an incubation time of 5 minutes, the pieces of fleece are centrifuged out and the a-galact-osidase activity of the filtrate determined with 40 mM
chlorophenol red galactoside solution (prepared according to European Patent Specification 110 0,146,866) at 5~8 nm.
There is obtained the standard curve illustrated in Fig. Z of the accompanying dra~nings c~rhich can be used for the examination of solutionswith unkno~,an T3 content, Example 4.
Preparation and cross-linking of rabbit I~;G-di~;oxi~enin c on~-j u~ate .
a) 3 g. rabbit I;G are dissolved in 300 mlo potassium phosphate buffer (l00 mrT, pH 8,5) and mixed with a solution of 58 mg. digoxigenin(3-succinidyl)-T~-hydroxy-succinimide in 30 ml. dioxan. After a reaction time of 2 hours, the protein solution is dialysed against a 200 fold volume of 20 mM potassium phosphate buffer (pIi 7.0) - 21 - ~ ,~
and adjusted via ultrafiltration (Amicon YM 100 F
membrane) to a concentration of 50 m~./ml.
For the cross-linking, to the batch are slowly added 35 ml, disuccinidyl suberate solution (concentration 10 mg.
disuccinidyl suberate/ml) dioxan). After completion of the cross-linking reaction, dialysis is carried out against a 500 fold volume of potassium phosphate buffer (50 mi~I, pH ~.2) and the fraction eluted on Superose 6 R in t he exclusion volume is separated off, stabilised with saccharose (concentration 6 m~, saccharose/mg. IgG) and lyophilised.
b) Dioxin test.
A fleece of 6Gg~ sulphite cellulose and 40g~ linters is impregnated to saturation with cross-linked rabbit IgG-di~oxioenin conjugate from Example 4a) (250 ~ ;./ml.
in 50 mT~I potassium phosphate buffer, pH 7.2) and dried at 50oC. for 60 minutes in a circulating air cabinet, In each case, 5 0 ~,,,.1. sheep anti-dioxin-IoG-~3-galactosidase conjugate (prepared analogously to the process described in J, Immunoassay, 4. 209-32/1983) (120 mU in phosphate-buffered physiological sodium chloride solution, 5 g./litre bovine serum albumin) are incubated for 5 minutes ~~rith 50 r.l. of a di~o;cin standard series and subsequently pipetted on to the above-prepared pieces of fleece (8 x 8 mm., thickness 0,5 mm.) of the diJoxin matrix. After an incubation time of 5 minutes, the pieces of fleece are, in each A
....
case, centrifuged off in a centrifuge and the ~i-galacto-sidase activity of the centrifugate determined with 40 ml~I
chloropheno~ red galactoside solution (prepared according to European Patent specification No, 0,146,866) at 578 nm.
The standard curve shown in Fig. 2 of the accomp-anying drawings is obtained, by means of which the unknown digoxin content in a solution can be determined.
Example 5 Impregnation of paper with thermally aggregated bovine serum albumin-streptavidin conjugate (tBSA-SA) and determination of the desorption rate 1. Impregnation 8 mm x 8 mm squares of paper (80 % polyester / 20 % cellulose /
20 % (referred to fibres) Etadurin) are impregnated with 42 ~cl of a solution of 0,5 ~g/~C1 tBSA-SA (Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutschland) in 50 mMol/1 potas-sium phosphate buffer, pH 7,0. The impregnated paper is dried for 30 minutes at 70 °C.
*trade mark 2. Determination of the desorption rate An impregnated fleece according to 1. is v_ortexed for 15 minutes in 1 ml of 50 mM potassium phosphate buffer, pH 7,0.
The supernatant is transferred to a 1 ml enzymun-plastic tube (Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutsch-land), which is internally precoated with 1 ~,g/ml tBSA-Biotin 1:1 (Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutschland) and incubated for one hour. After washing twice with water 1 ml of a solution of peroxidase-biotin-conjugate (200 mU/ml, Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutschland) is added into the tube and is incubated there for 30 minutes. After washing twice with water 2,2'-azino-di-[3-ethylbenzthiazolinsulfonat(6)] is added into the tube and the reaction solution is measured at 405 nm. The system is cali-brated with a set of tBSA-SA-solutions of known concentration.
When using the impregnated paper according to 1. there are desorbed 18 ng tBSA-SA, which is equal to 0,09 %. When omitting the drying step in the impregnation procedure according to 1.
there are desorbed 560 ng tBSA-SA, which is equivalent to 2,7 %. It has to be concluded from that, that protein immobi-lized according to the present invention is essentially firmer bound to the solid phase than the solely adsorbed protein.
A
' ~ , The patent specifications referred to herein are more fully identified below.
European Patent Specification 0 274 911, P.J. Degen et al, published July 20, 1988, Pall Corporation;
Federal Republic of Germany Offenlegungsschrift 3 446 636, H. Jering et al, laid open June 26, 1986, Boehringer Mannheim GmbH;
U.K. Patent Specification 1 505 400, W.F
Barg, Jr., published March 30, 1978, American Cyanamid Company;
European Patent Specification 0 122 209, M. Delsage et al, published October 17, 1984, Immunotech, S.A.;
European Patent Specification 0 269 092, W Tischer et al, published June 1, 1988, Boehringer Mannheim GmbH;
European Patent Specification 0 146 866, M. Kuhr et al, published July 3, 1985, Boehringer Mannheim GmbH.
A
For the cross-linkin~, to the batch are slo~-~ly added 35 ml, disuccinidyl suberate (concentration ZO mg, di-succinidyl suberate/ml. dioxan). After completion of the cross-linking reaction, dialysis is carried out against a 500 fold volume of potassium phosphate buffer (50 mM, pH 7.2) and the fraction eluted on Superose 6 '~ in the exclusion volume separated off, stabilised with saccharose (concentration 6 mg. saccharose/mg. IgG) and lyophilised.
b) Triiodothyronine (T3) test.
A fleece of 605 sulphite cellulose and 40~' linters is impregnated with cross-linked rabbit IgG-T3 conjugate from Example 3a) in a concentration of 250 ~.o./ml. in A
50 mP~I potassium phosphate buffer (pH 7.2) and, after complete saturation, dried at 50oC, for 60 minutes in a circulating air cabinet. In each case, 50 f.,~.l~ of sheep anti-T3-IgG-(3-galactosidase conjugate (prepared analogously to the process described in J. Immunoassay, 4, 209-32'7/
2983)(120 mU in phosphate-buffered physiological sodium chloride solution, 5 g./litre bovine serum albumin) are incubated for 5,minutes with 50 ~,1, of a T3 standard series and subsequently pipetted on to the above-produced pieces of fleece (8 x 8 mm., thickness 0.5 mm,) of the T3 matrix, After an incubation time of 5 minutes, the pieces of fleece are centrifuged out and the a-galact-osidase activity of the filtrate determined with 40 mM
chlorophenol red galactoside solution (prepared according to European Patent Specification 110 0,146,866) at 5~8 nm.
There is obtained the standard curve illustrated in Fig. Z of the accompanying dra~nings c~rhich can be used for the examination of solutionswith unkno~,an T3 content, Example 4.
Preparation and cross-linking of rabbit I~;G-di~;oxi~enin c on~-j u~ate .
a) 3 g. rabbit I;G are dissolved in 300 mlo potassium phosphate buffer (l00 mrT, pH 8,5) and mixed with a solution of 58 mg. digoxigenin(3-succinidyl)-T~-hydroxy-succinimide in 30 ml. dioxan. After a reaction time of 2 hours, the protein solution is dialysed against a 200 fold volume of 20 mM potassium phosphate buffer (pIi 7.0) - 21 - ~ ,~
and adjusted via ultrafiltration (Amicon YM 100 F
membrane) to a concentration of 50 m~./ml.
For the cross-linking, to the batch are slowly added 35 ml, disuccinidyl suberate solution (concentration 10 mg.
disuccinidyl suberate/ml) dioxan). After completion of the cross-linking reaction, dialysis is carried out against a 500 fold volume of potassium phosphate buffer (50 mi~I, pH ~.2) and the fraction eluted on Superose 6 R in t he exclusion volume is separated off, stabilised with saccharose (concentration 6 m~, saccharose/mg. IgG) and lyophilised.
b) Dioxin test.
A fleece of 6Gg~ sulphite cellulose and 40g~ linters is impregnated to saturation with cross-linked rabbit IgG-di~oxioenin conjugate from Example 4a) (250 ~ ;./ml.
in 50 mT~I potassium phosphate buffer, pH 7.2) and dried at 50oC. for 60 minutes in a circulating air cabinet, In each case, 5 0 ~,,,.1. sheep anti-dioxin-IoG-~3-galactosidase conjugate (prepared analogously to the process described in J, Immunoassay, 4. 209-32/1983) (120 mU in phosphate-buffered physiological sodium chloride solution, 5 g./litre bovine serum albumin) are incubated for 5 minutes ~~rith 50 r.l. of a di~o;cin standard series and subsequently pipetted on to the above-prepared pieces of fleece (8 x 8 mm., thickness 0,5 mm.) of the diJoxin matrix. After an incubation time of 5 minutes, the pieces of fleece are, in each A
....
case, centrifuged off in a centrifuge and the ~i-galacto-sidase activity of the centrifugate determined with 40 ml~I
chloropheno~ red galactoside solution (prepared according to European Patent specification No, 0,146,866) at 578 nm.
The standard curve shown in Fig. 2 of the accomp-anying drawings is obtained, by means of which the unknown digoxin content in a solution can be determined.
Example 5 Impregnation of paper with thermally aggregated bovine serum albumin-streptavidin conjugate (tBSA-SA) and determination of the desorption rate 1. Impregnation 8 mm x 8 mm squares of paper (80 % polyester / 20 % cellulose /
20 % (referred to fibres) Etadurin) are impregnated with 42 ~cl of a solution of 0,5 ~g/~C1 tBSA-SA (Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutschland) in 50 mMol/1 potas-sium phosphate buffer, pH 7,0. The impregnated paper is dried for 30 minutes at 70 °C.
*trade mark 2. Determination of the desorption rate An impregnated fleece according to 1. is v_ortexed for 15 minutes in 1 ml of 50 mM potassium phosphate buffer, pH 7,0.
The supernatant is transferred to a 1 ml enzymun-plastic tube (Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutsch-land), which is internally precoated with 1 ~,g/ml tBSA-Biotin 1:1 (Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutschland) and incubated for one hour. After washing twice with water 1 ml of a solution of peroxidase-biotin-conjugate (200 mU/ml, Boehringer Mannheim GmbH, Mannheim, Bundesrepublik Deutschland) is added into the tube and is incubated there for 30 minutes. After washing twice with water 2,2'-azino-di-[3-ethylbenzthiazolinsulfonat(6)] is added into the tube and the reaction solution is measured at 405 nm. The system is cali-brated with a set of tBSA-SA-solutions of known concentration.
When using the impregnated paper according to 1. there are desorbed 18 ng tBSA-SA, which is equal to 0,09 %. When omitting the drying step in the impregnation procedure according to 1.
there are desorbed 560 ng tBSA-SA, which is equivalent to 2,7 %. It has to be concluded from that, that protein immobi-lized according to the present invention is essentially firmer bound to the solid phase than the solely adsorbed protein.
A
' ~ , The patent specifications referred to herein are more fully identified below.
European Patent Specification 0 274 911, P.J. Degen et al, published July 20, 1988, Pall Corporation;
Federal Republic of Germany Offenlegungsschrift 3 446 636, H. Jering et al, laid open June 26, 1986, Boehringer Mannheim GmbH;
U.K. Patent Specification 1 505 400, W.F
Barg, Jr., published March 30, 1978, American Cyanamid Company;
European Patent Specification 0 122 209, M. Delsage et al, published October 17, 1984, Immunotech, S.A.;
European Patent Specification 0 269 092, W Tischer et al, published June 1, 1988, Boehringer Mannheim GmbH;
European Patent Specification 0 146 866, M. Kuhr et al, published July 3, 1985, Boehringer Mannheim GmbH.
A
Claims (12)
1. A process for immobilizing a protein containing substance onto a solid phase comprising:
(i) forming a water soluble aggregate of a polymer containing more than one molecule of a particular protein;
(ii) contacting a liquid containing said aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said aggregate immobilized and non-covalently bound thereon.
(i) forming a water soluble aggregate of a polymer containing more than one molecule of a particular protein;
(ii) contacting a liquid containing said aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said aggregate immobilized and non-covalently bound thereon.
2. A process of claim 1, wherein said forming of said water soluble aggregate comprises mixing more than one molecule of a particular protein with a polyfunctional linker.
3. A process of claim 1 or 2, wherein said drying comprises treating said solid phase to contain less than 7% moisture by weight at 20°C and 50%
relative humidity.
relative humidity.
4. A process of claim 1 or 2, wherein, after contacting of the solid phase with the liquid containing said aggregate, in step (ii), no washing step takes place prior to step (iii).
5. A process of claim 3, wherein, after contacting of the solid phase with the liquid containing said aggregate, in step (ii), no washing step takes place prior to step (iii).
6. A process for immobilizing a protein containing substance onto a solid phase comprising:
(i) forming a water soluble protein homopolymer aggregate;
(ii) contacting a liquid containing said protein homopolymer aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said protein homopolymer aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said protein homopolymer aggregate immobilized and non-covalently bound thereon.
(i) forming a water soluble protein homopolymer aggregate;
(ii) contacting a liquid containing said protein homopolymer aggregate in dissolved form with a hydrophilic solid phase so as to adsorptively, and non-covalently bind said protein homopolymer aggregate to said hydrophilic solid phase; and (iii) drying said solid phase to form a solid phase having said protein homopolymer aggregate immobilized and non-covalently bound thereon.
7. A solid phase having a composite comprising protein aggregates bound thereto, comprising:
a hydrophilic solid phase having non-covalently bound thereto a protein aggregate wherein said aggregate is formed by aggregating more than one molecule of a particular protein to form a polymer containing aggregate prior to contacting said aggregate with said solid phase, wherein said solid phase is dried following contact of said solid phase with said polymer containing aggregate.
a hydrophilic solid phase having non-covalently bound thereto a protein aggregate wherein said aggregate is formed by aggregating more than one molecule of a particular protein to form a polymer containing aggregate prior to contacting said aggregate with said solid phase, wherein said solid phase is dried following contact of said solid phase with said polymer containing aggregate.
8. A solid phase of claim 7, wherein said solid phase is characterized by a moisture content of less than 7% by weight at 20°C and 50% relative humidity.
9. A solid phase having a composite comprising protein aggregates bound thereto, comprising:
a hydrophilic solid phase having non-covalently bound thereto a protein homopolymer aggregate, wherein said homopolymer aggregate is formed prior to contacting said homopolymer aggregate to said solid phase, wherein said solid phase is dried following contact of said solid phase with said homopolymer aggregate.
a hydrophilic solid phase having non-covalently bound thereto a protein homopolymer aggregate, wherein said homopolymer aggregate is formed prior to contacting said homopolymer aggregate to said solid phase, wherein said solid phase is dried following contact of said solid phase with said homopolymer aggregate.
10. A solid phase having a composite comprising protein aggregates bound thereto comprising a hydrophilic solid phase having non-covalently bound thereto a protein aggregate, wherein said aggregate is formed by mixing more than one molecule of a particular protein with a polyfunctional linker to form a protein containing aggregate prior to contacting said protein aggregate with said hydrophilic solid phase, wherein said solid phase is dried following contact of said solid phase with said protein aggregate.
11. A method for determining an analyte in a liquid sample, comprising contacting said liquid sample with the protein aggregate containing solid phase of claim 7, 8, 9 or 10, wherein said aggregate comprises a substance which binds with said analyte and determining said analyte bound to said aggregate.
12. Use of a solid phase according to claim 7, 8, 9 or 10, for an analytical determination.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3842700A DE3842700A1 (en) | 1988-12-19 | 1988-12-19 | METHOD FOR PROTEIN IMMOBILIZATION ON A SOLID PHASE, PROTEIN-CARRYING SOLID PHASE PRODUCED THEREOF AND THE USE THEREOF |
| DEP3842700.1 | 1988-12-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2005926A1 CA2005926A1 (en) | 1990-06-19 |
| CA2005926C true CA2005926C (en) | 1999-11-16 |
Family
ID=6369520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002005926A Expired - Lifetime CA2005926C (en) | 1988-12-19 | 1989-12-19 | Process for immobilising a protein on a solid phase |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5310885A (en) |
| EP (1) | EP0374778B1 (en) |
| JP (1) | JPH087213B2 (en) |
| AT (1) | ATE103936T1 (en) |
| AU (1) | AU607088B2 (en) |
| CA (1) | CA2005926C (en) |
| DE (2) | DE3842700A1 (en) |
| ES (1) | ES2051977T3 (en) |
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-
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- 1989-12-12 AU AU46165/89A patent/AU607088B2/en not_active Ceased
- 1989-12-16 ES ES89123321T patent/ES2051977T3/en not_active Expired - Lifetime
- 1989-12-16 EP EP89123321A patent/EP0374778B1/en not_active Expired - Lifetime
- 1989-12-16 DE DE89123321T patent/DE58907404D1/en not_active Expired - Lifetime
- 1989-12-16 AT AT89123321T patent/ATE103936T1/en not_active IP Right Cessation
- 1989-12-19 JP JP1327506A patent/JPH087213B2/en not_active Expired - Lifetime
- 1989-12-19 CA CA002005926A patent/CA2005926C/en not_active Expired - Lifetime
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1992
- 1992-05-19 US US07/887,239 patent/US5310885A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP0374778A2 (en) | 1990-06-27 |
| JPH087213B2 (en) | 1996-01-29 |
| ES2051977T3 (en) | 1994-07-01 |
| US5310885A (en) | 1994-05-10 |
| ATE103936T1 (en) | 1994-04-15 |
| AU4616589A (en) | 1990-06-21 |
| DE58907404D1 (en) | 1994-05-11 |
| EP0374778B1 (en) | 1994-04-06 |
| AU607088B2 (en) | 1991-02-21 |
| DE3842700A1 (en) | 1990-06-21 |
| JPH02262600A (en) | 1990-10-25 |
| EP0374778A3 (en) | 1991-10-09 |
| CA2005926A1 (en) | 1990-06-19 |
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