SUSPENSION OF PARTICLES WITH A DIAMETER OF LESS THAN 1 mu M WITH IMMOBILIZED ENZYME AND PROTEIN

Abstract: The invention relates to a new type of particle suspension, which contains at least one immobilised enzyme and at least one other immobilised protein or a protein derivative, to be used in the determination of a cell, a virus or another component in a sample and the use of the suspension in clinical, histochemical or microscopical application, as well as in other diagnostic application areas.

Description: Suspension of particles with a diameter of less than 1 urn with immobilized enzyme and protein . The present invention relates to a new type of enzyme protein conjugate to be used *• in the determination of a cell, a virus or another component in a sample and the use of the conjugate in clinical, histochemical or microscopical application as well as in other diagnostical applications. Chemical analysis based on specific biological affinity between receptor and ligand, as for example between antibody and antigen, has been used for several years in the separation and determination of cells, virus or other components (which are called analytes below) in samples. Examples of this are analysis based on antibodies, i.e. the immunotechniques. Antibodies or other proteins are here used for the specific binding of the analyte (which can be an antibody) in the sample. Detection of antibody-ligand complex or of protein-analyte complex or of free antibody or analyte can be done in several ways. In the so called radioimmunoassay, RIA, radioactively marked antibodies, ligands or proteins are used, but also for example fluorescent or luminiscent groups, different enzymes and particles have been used as markers in immunoassays (see e.g. Ngo and Lenhoff, editors, Enzyme-mediated immunoassay, pages 3-32, Plenum Press, 1985, ref. 1). RIA has however dominated. In spite of the advantages of RIA, disadvantages as for example the instability of several gamma-emitters and the health hazards by the synthesis and the handling of the radioactive markers have lead to the gradual replacement of the radioactive markers by other types of markers. Especially enzyme immunoassay (El A; ref. 1 and Engvall, Meth. Enzymol., 70, pages 419-439; ref. 2) has turned out to have advantages and is more and more used. EIA is divided into homogeneous and heterogeneous EIA. Heterogeneous EIA, which includes ELISA (enzyme linked im unosorbent assay) is based on the same principles as RIA and very often gives similar sensitivity and specificity. These EIA techniques require methods for the conjugation of antibody or another protein to a suitable enzyme. Even if several such methods based on chemical reagents are described in the literature (for example glutaraldehyde, maleiimides, and other bifunctional molecules, periodate or thiol substances; see O ' Sullivan and SUBSTITUTE SHEET ISA/SE Marks Meth. Enzymol. vol. 73, pages 147-166, ref. 3, ref. 2 and other relevant articles in Meth. Enzymol., volumes 70, 73 and 92), they all have their drawbacks. It is for instance difficult with existing techniques to produce, reproducible and controllable, for example enzyme-antigen, enzyme-antibody or enzyme-protein A and other enzyme-protein conjugates. One obtains conjugates which are heterogeneous in size and structure. Furthermore, conjugation may cause decreased enzyme activity or specificity of for example the antibody. Moreover, even if the sensitivity often is sufficient, EIA is in many cases limited by insufficient sensitivity. One has tried to increase the sensitivity by using more or less complicated multi-component systems such as streptavidin-biotin or avidin- biotin, which both favours the formation of oligomeric conjugates when biotin labelled substances are used (Kendall et al- J. Immunol. Meth., vol. 56, page 329, 1983, ref. 4, see also ref. 1-3 and relevant articles in Meth. Enzymol.) or so called enzyme-cascades (Self, US Patent Application No. 307, 600; ref. 5). Other systems utilise soluble, oligomeric enzyme-conjugates described e.g. by Leary et al, Proc. Nat. Acad. Sci., vol 80, page 4045, 1983; ref.6). Even if these so called amplification techniques are useful in several applications, they have their limitations and disadvantages and Ngo mentions in a review that increased sensitivity of EIA and better methods for the preparation of enzyme- ligand or enzyme-receptor conjugates are desirable. One of the objects of this invention is to reduce these disadvantages by EIA and an enzyme-antibody or in general enzyme-protein conjugate of high activity is described. Moreover, it is described how this reagent can be used for the detection of a cell, a virus or another component in a sample and how an increased sensitivity compared to earlier methods can be obtained by using the conjugate according to the invention. These and other aims can be reached, according to the invention, by covalent or noncovalent binding of enzyme and antibody, or enzyme and streptavidin, or enzyme and antigen (or in general, enzyme and protein, or a fragment or a derivative thereof) to insoluble particles, which have a size of less than 1 μm and which have a similar density as water and which form a stable suspension in the media - usually buffered water - used in the konjugation reaction to the particles and in the determination of the analyte (usually buffered water), and, in addition, by employing so formed particle-conjugate suspension instead of and in the same way as previously described enzyme conjugates, for the specific detection of the analyte. This facilitates, according to the invention, the preparation of enzyme conjugates which form stable particle suspensions in buffered water and which can be used in the same way as previously used conjugates and which give high sensitivity when used in different analytical applications. The particles can consist of a polymer substance which can consist of natural, semisynthetic or synthetic materials. As examples one can mention plastics (polyvinylalcohol, polystyrene, etc.), copolymers (e.g. of acrylic acid and styrene) cross-linked polysaccharides, etc. The particles can be compact or porous, the latter allowing higher amount of bound enzyme and a higher enzyme activity of the particle suspension. Immobilization of the enzyme and protein to the particle suspension can easily be carried out with different methods. The surface of the particles can be more or less hydrophobic. A hydrophobic surface can be used for noncovalent binding of enzyme and ligand or receptor to the particle. Both hydrophobic (for example polystyrene particles, which can be nitrated, reduced and diazotized) and hydrophilic surfaces can be chemically modified to introduce reactive groups for covalent binding of proteins (see for instance Methods Enzymology volumes 44, 104 and 135.) Particles modified with for example cyanate, tosyl or tresyl groups, imidoesters, other esters, aldehyde, epoxi, divinylsulphone groups can be prepared by reaction with CNBr, tosyl chloride, tresyl chloride, different carbodiimides, periodate, and other reagents. After this so called activation step, the so called immobilisation step or coupling of the enzyme and the protein, simultaneously or consecutively, to the particle is carried out. The reaction in these examples takes place between nucleophilic groups (amines, thiols) on the enzyme and the protein and the reactive groups on the particles. Alternatively, the enzyme and the protein is activated with for example periodate or carbodiimide, instead of activation of the particle. Here, the enzyme can be bound first and thereafter the antibody or the protein. In addition, antibodies can be bound via their thiol groups or via a smaller fragment of the antibody. To achieve this, thiol reactive reagents can be used, alternatively, one can, before the immobilisation of the antibody, immobilise for example protein A or a secondary antibody to the antibody or the protein, simultaneously or after immobilisation of the enzyme, and thereafter the resulting particle suspension can be mixed with the desired antibody or protein. After the immobilisation, performed at suitable pH and temperature, the particle suspension is washed after centrifugation or filtration, and the particle suspension can thereafter be used in, for example, those types of EIA mentioned above. The concentration of the particles, reagents for activation, enzyme and protein in the coupling step is kept at a suitable level to avoid crosslinked particles. The so prepared particle suspension with bound enzyme and protein, can eventually be size separated using e.g a Sephadex-column. The ratio of enzyme to protein is chosen according to the application. A high ratio of enzyme to receptor or ligand can give a high enzyme activity but can result in a low binding capacity. A person skilled in the art can decide the optimal conditions for a given situation. Eventually the enzyme can be coupled first and then the ligand or the receptor can be coupled either directly on the remaining particle surface or on the enzyme with the help of crosslinker, e.g glutaraldehyde or maleiimide, or with the help of a reagent of the type periodate (which can be used to oxidize carbohydrate, on for example peroxidase, to aldehyde groups which are reactive towards the amino groups of antibodies or proteins). One can also according to the invention, couple a covalent enzyme conjugate, for example a soluble monoconjugate between enzyme and protein, or a noncovalent conjugate between enzyme and protein, for example PAP (peroxidase- antiperoxidase conjugate) to the particles The choice of enzyme and enzyme substrate can easily be done by a person skilled in the art and does not limit the scope of the invention. All enzymes, which can be coupled to the particles, which are used in the invention, can be used according to the invention. Any enzyme and substrate, which are suitable for the application can be used, and the same enzymes and substrates which have been used in immunohistochemical studies, immunoblotting, microscopical studies an in ELISA can be used. Examples of enzymes which can be used are peroxidase, alkaline phosphatase, galactosidase, urease, glucose-6-phosphate dehydrogenase and luciferase. To increase the enzyme activity per particle, an enzyme with high turnover number and relatively low molecular weight can be used. Examples of enzyme substrates which can be used together with the reagent according to the invention, are given in references 1-13 and in relevant articles in Meth. Enzymol. volumes 70, 73 and 92. As mentioned above, the protein, which is coupled to the particle suspension together with the enzyme, shall be able to biospecifically bind the analyte (that is the component which is to be detected in the sample) or an antibody, another protein, or another type of molecule which is used in the analytical procedure. The protein, which is coupled to the particle suspension together with the enzyme, can for example an antibody, protein A, streptavidin, a lectin, avidin or another type of protein or a derivative thereof, or a recombinant protein or antibody, or a fragment thereof.be identical with the analyte or be an analogue thereof. This substance can for instance be an antibody, a lectin, avidin, another sort of protein or glycoprotein, a carbohydrate derivative, a glycolipid, a neoglycoprotein, a steroid derivative, a coenzyme derivative, a metabolite derivative, an analogue or metabolite of a pharmaceutical preparation, a metabolite, hormone, nucleotide or a derivative thereof. Examples of applications of the reagent according to the invention are in ELISA (ref. 1 and 2), in immunohistochemical studies (Avrameas, Histochem. J. vol. 4, page 321, 1972; ref. 7), in immunoblotting (Tsang et al, Meth. Enzymol. vol. 92, page 377; ref. 8), for the detection of antibodies in cell cultures etc. The sample, which contains the analyte, can be in the form of a fluid (tears, saliva, serum, urine, water sample, etc.) in the form of more or less solid material (tissue, nitrocellulose etc.). The cells, virus or components which can be analyzed with the particle suspension according to the invention, are for example pathogenic organisms such as parasites, yeast cells, bacteria, mycoplasma, virus, toxins, proteins, enzymes, antibodies and other proteins, pharmaceutical preparations and their metabolites, other metabolites, hormones, steroids, prostaglandins, nucleotides and biomolecules in cells, virus, on cell surfaces, in tissues or in the circulation and other analytes, for instance in waste water, earth, plants, animals and food. The solid phase used for the separation step in ELISA with the particle suspension according to the invention, can be of the same type that have been used in ELISA with earlier described enzyme conjugates (see e.g. pages 388-391 in Meth. Enzymol. vol. 70, 1980), such as plastics in the form of test-tubes, microtiter plates, particles, filters, etc., glass fiber orfilters of paper, ion exchangers, agarose particles, Sephadex particles, polyacrylamid gel, bentonite, magnetic preparations of cellulose, agarose or plastics, etc. The shape of the solid phase depends on the application and for instance beads, columns, dipsticks, microtiterplates, membranes, filters or test-tubes can be used. After separation of the bound particle conjugate from the non-bound fraction, the activity of either the bound or non-bound fraction is determined. Examples of specific applications, where the particle suspension according to the invention can be used, are competitive and noncompetitive ELISA-methods (Meth. Enzymol. vol. 73, page 383 Hsu et al; ref. 9). The enzyme conjugates that have been used earlier in these methods are consequently replaced with the particle suspension according to the present invention. Otherwise, the techniques when carrying out the assays are identical or similar with the in literature described EIA- techniques (ref. 1.2 etc.). Some examples of ELISA principles with which the particle suspension according to the invention can be used are shown in the annexed schemes, where L is the analyte, that is the component which is to be determined, A is antibody or protein or another component with specific affinity to L, B is antibody, protein A, or another protein or another component with specific affinity to L or to A, E is enzyme, S is enzyme substrate and P is product, which is detected, Ln-O-Em, An-O-Em, and Bn-O-Em symbolise examples of particles in the particle suspension according to the invention, -O- is the particle and / is the solid phase: Competitive type of ELISA L ► L:Ab n -O-E„, An-O-Em S /-L ► /-L:An-O-Em P Noncompetitive (sandwich; A = B. or A andB are different) /-A + L > /-A:L S An-O-Em < - — » /-A:L:An-O-Em Indirect (A = analyte) /-L + A -→ l-L'.A S Bn-O-Em -" * — ► /-L:A:Bn-O-Em P Protein A or G, which both specifically binds to certain types of antibodies, can in the last scheme above be bound to the particles of the particle suspension and resulting particle suspension is used in the same manner as previous enzyme- protein A conjugates. In addition, peroxidase-antiperoxidase conjugate (PAP- conjugate) can be bound to the particles of the particle suspension and the resulting particle conjugate suspension can be used in the same manner as previous PAP- conjugates. In references 1 and 2 given above (and references given in these articles) the ELISA-techniques with soluble enzyme conjugates are explained extensively, as well as the buffers and other reagents involved and the applications, and the techniques and the applications when using the particle suspension according to the invention are similar or identical. The particle suspension according to the present invention can also be used together with many of the amplification techniques described for ELISA (avidin-biotin, , streptavidin-biotin, enzyme-cascade, USERIA etc.). Thus, as an example, streptavidin-biotin, avidin-biotin, or avidin (A) and enzyme (E) can be coupled to the particles ( -O- ) and the resulting particle conjugate suspension can be used for the detection of bound biotin-labelled antibody or analyte and, for example, the final complexes shown in the annexed scheme can be detected with the suspension as shown below (streptavidin can be used instead of avidin): S /-A:L-biotin:avidin n -O-Em ^ P S /-A:L-biotin:avidin:biotin n -O-Em /-A:L:A:A-biotin:avidin n -O-E m (etc; Dakopatts Product list, 1988; ref 10) Review articles on avidin-biotin, see HSU et al page 467 in Ngo and Lenhoff (ref 1) and Wilchek and Bayer, Immunology Today, vol 5, page 39, 1984 (ref 1 1) The sensitivity can be increased, by further reacting the product P, formed in the above enzyme reactions with other enzymes (according to for example ref. 5 above) or by transforming P to a luminiscent substance according to for example Wannlund and Deluca, Meth. Enzymol. vol. 92, page 426 (ref. 12). The final product is preferably detected with conventional ELISA-technique, that is by the eye, by measuring the absorbance, fluorescens, etc. or the reaction is followed kinetically (Tsang et al, Meth. Enzymol. vol. 92, page 391; ref. 13). Furthermore, the product P can be radioactive and can be separated from the substrate with for example a Sephadex-column and be quantified with a scintillation counter (USERIA; ref. 9 above). The above constitutes only examples of how the invention may be used in actual practice and are not intended to limit the scope of the invention. Some examples of how the invention can be applied are described in the Examples below, which in no way are intended to limit the scope of the invention. EXAMPLES . Several different types of plastic particles that form stable suspensions can be used but the examples below exemplify the use of particles (below called latex particles) made by copolymerisation of acrylic acid and scyrene using emulsion polymerisation. Also, polystyrene particles belong to this category. Centrifugation is used in the examples below to remove unbound reagent but can be substituted for by e.g. filtration methods. The relative amounts of particles and their size, enzymes, proteins, buffers, reagents, pH, are typical but can be varied by the biochemist/chemist to obtain suitable conjugates for the application.Polymer particles e.g.of the type in Example l.E can also be used. 1 . A. Peroxidase-latex-antibody conjugate prepared via periodate activation of peroxidase carbohydrate . 1. Horseradish peroxidase (Boehringer; 16 mg; 250 ϋ/mg) was dissolved in 0.1 M NaHC0 3 (pH 8.1) and 1 ml of 8 mM NaI0 4 was added at room temperature and allowed to react for 2 h in the dar . 2. An amino group containing particle preparation (15 mg dry weight of particles; 0.1 μm average particle size, made by reaction of hexane diamine with EDC and carboxyl group containing particles, called CML; Seradyn Inc. USA) was suspended in 1 ml of 0.1 M NaHCC>3, pH 9.0, (hereinafter called buffer D) was added to the oxidized peroxidase and the mixture was dialyzed twice against 600 ml buffer D. 3. After two days at 4 °C, NaCNBH 3 (450 μl; 12 mg/ml of buffer D) was added to the mixture and allowed to react for 2 h at room temperature . . The suspension was centrifuged at a suitable speed for 15 minutes at 15 °C, the supernatant discarded and 6 ml of buffer D added to the soft pellet of particles. The centrifugation procedure was repeated once, and was followed by addition of 6 ml MOPS-HC1 (3- (N-morpholino) propanesulfonic acid-HCl) (50 mM; pH 7.5; hereinafter called buffer E) , centrifugation, removal of the supernatant, addition of 6 ml of Buffer E and centrifugation. The suspension was diluted to 1 ml. 5. Rabbit antibodies to human alfa-1-fetoprotein (immunoglobulins, 0.5 mg; Dakopatts, Denmark), dissolved in 0.6 ml buffer E, was added to the suspension. 6. EDC (l-ethyl-3- (3-dimethyl aminopropyl) -carbodiimide; 0.5 mg dissolved in 40 μl distilled water) was added, and the reaction mixture was allowed to stand overnight at 4 °C. 7. The suspension was washed by repeated centrifugation (three times with buffer E) and the soft pellet was finally diluted to 1 ml with buffer E and stored at 4 °C. The particle suspension according to the invention may also be prepared by coupling of the antibody to the aldehyde groups (obtained by periodate oxidation) on the peroxidase. In that case EDC can be omitted, and a borohydride is used for final reduction of Schiff bases (formed between antibody amino groups and peroxidase aldehyde groups) and remaining free aldehyde groups . Another possibility to bind enzyme and protein to the particle suspension is to form diazonium groups on polystyrene particles via nitration, reduction and diazotisation. Such diazonium group containing particles are very reactive against hydrophobic groups on enzymes and proteins, antibodies (e.g. at pH 7-9) . In this latter method no preactivation of the enzyme or protein is required, and enzyme and protein are added to the activated particles simultaneously or after each other. Second, enzyme and protein can also be coupled directly (without prior modification of the enzyme or protein) to activated particles containing active esters like tosylate, tresylate or cyanate (obtained by e.g. reaction of hydroxyl group containing particles with tosyl chloride, tresyl chloride or CNBr, respectively) . Third, carboxyl group containing particles can be activated with EDC and the so activated particles are suitable for binding of enzyme and protein without prior modification of enzyme or protein. Example l.D below is only one example of such a procedure. On the market,there are plenty of polystyrene based latex particles or hydroxyl group or carboxyl group containing particles which are suitable for activation via diazonium or ester or carbodiimide groups, respectively. Instead of activating the carbohydrate on the enzyme (e.g. peroxidase) as in Example 1A, one can activate the carboxyl groups on the enzyme and/or protein with a suitable reagent of which there are many examples in the literature. Below is an example of such a procedure. 1 . B . 1. In an alternative procedure to I.A., 1 mg peroxidase in which the carboxyl groups of the enzyme were activated with EDC (such enzyme preparations are commercially available, e.g. Immunopure^ activated peroxidase, Pierce, Rockford, USA) was dissolved in 1 ml cold, distilled water and added to the same type of amino group containing particle preparation (7.5 mg dry weight) as described above (see 1.A.2) dissolved in 1 ml of buffer D. The mixture was allowed to react overnight at room temperature. Remaining reactive groups on the peroxidase were blocked by addition of 0.2 M Tris-HCl, pH 9.0, to the reaction mixture. 2. After 2 h at room temperature, the suspension was washed by repeated centrifugation (four times with buffer E) . 3. Rabbit antibodies to human alfa-1-fetoprotein (immunoglobulins; 1.2 mg; dissolved in 600 μl buffer E) was added to the suspension (diluted to 1 ml with buffer E) . The mixture was then treated with EDC, centrifuged and the suspension stored as described in l.A (steps 6 and 7) . l . C. Preparation of peroxidase-latex-protein A conjugate The preparation of this conjugate was as for l.A (or l.B), except that protein A (400 μg; dissolved in 0.7 ml buffer E) from Staphylococcus aureus was added instead of the rabbit antibody in 1.B.3. l . D . Preparation of PAP-latex conjugate 1. PAP (a noncovalent, soluble conjugate between horseradish peroxidase and rabbit antibody to horseradish peroxidase; Dakopatts, Denmark; 1.2 mg; dissolved in 920 μl buffer E) was added to 50 μl of a latex particle suspension (carboxyl group containing particles, CML from Seradyn Inc. USA; 5 mg dry weight; average diameter 0.2 μm) . The mixture was allowed to stand for 45 minutes at ambient temperature. 2. EDC (see l.A.6; 0.3 mg dissolved in 30 μl distilled water) was added and the mixture allowed to stand for 1 h at room temperature, after which the suspension was washed by centrifugation with buffer E as described in l.A.7. I . E . Preparation of PAP-nanoparticle conjugate These particles were prepared with a analogous procedure to the procedure in l.D, employing carboxyl group containing plastic particles of 400 Angstrom average diameter (Rhone-Poulence) . The suspension was washed via dialysis against buffer. 2. Enzyme immunoassay The examples below exemplify the application of particle suspensions in microtiter plate assays. Other conditions for the assays as well as numerous other applications are possible. 2. A and 2. B . Test of conjugat es l . A and l . B . 1. A microtiter plate was coated with rabbit antibodies to human alfa-1-fetoprotein (immunoglobulins; Dakopatts, Denmark; 200 μl in buffer A; 10 mM sodium phosphate + 145 mM NaCl, pH 7.2) overnight at 4 °C. 2. The plate was washed with buffer A containing 0.1 % Tween 20 and 0.5 M NaCl (hereinafter called buffer B) . 3. Dilutions in buffer B of human alfa-l-fetoprotein (Dakopatts, Denmark; 0.1 ml in each well) was added and incubated for 2 h at room temperature . 4. As step 2. 5. Conjugates l.A (or l.B) was added (0.1 ml in each well; diluted 10-100 times with buffer B) and incubated for 1 h at room temperature. 6. As step 2. 7. Substrate solution (100 μl of a solution of 8 mg 1,2- phenylene diamine dihydrochloride dissolved in 12 ml of 0.1 M citric acid-phosphate buffer, pH 5.0, and 12 μl of 30% H2O2; hereinafter called buffer C) was added and after 30 minutes the absorbances of the various wells were measured at 490 nm with a multiscanning spectrophotometer. 2 . C . Test of conjugate l . C. 1. A microtiter plate was coated overnight at 4 °C with human alfa-1-fetoprotein (200 μl; 2 μg per ml of buffer A) . 2. The plate was washed three times with buffer B. 3. Dilutions (100 μl) of rabbit antibodies (immunoglobulins) to human alfa-1-fetoprotein (Dakopatts, Denmark; antibodies diluted in buffer B) was added to the wells of the microtiter plate. Incubation for 2 h at room temperature. 4.The plate was washed as in step 2. 5.Conjugate l.C (100 μl; properly diluted with buffer B) was added to the wells of the plate, which was incubated for 1 h at room temperature . 6. The plate was washed as in step 2. 7. Substrate solution was added as in step 7 of 2.A, and after 30 minutes the absorbances of the various wells were measured at 490 nm with a multiscanning spectrophotometer. 2 . D . Test of the PAP-particle latex conjugates l . D and I . E . These conjugate were tested as described in 2.C. until step 4, after which the following procedure was used. 5. Swine antibodies to rabbit immunoglobulins (Dakopatts, Denmark) was added (0.5 mg and 100 μl in each well) and was incubated for 1 h at room temperature. 6. The plate was washed with buffer B three times. 7. Conjugate l.D (diluted 20 times with buffer B; 100 μl in each well) was incubated for 1 h at room temperature. 8. See step 2 in 2.B. 9. See 2.B.7. Conjugate l.E was tested according to the same principle (i.e. conjugate I.E. in step 7 was used instead of conjugate l.D) . The particle suspensions above gave as high or higher sensitivity than the corresponding soluble conjugates (commercially available from Sigma, peroxidase-protein A or from Dakopatts, PAP and peroxidase-antibody conjugates) .

Claims: CLAIMS 1. Particle suspension, characterised by that the particles contain immobilised enzyme and at least one other immobilised protein or a protein derivative, and by that the size of the particles are below 1 μm. 2. A particle suspension according to claim 1, in which the protein is an antibody, protein A, streptavidin, avidin, another protein or a fragment or a derivative thereof. 3. Use of the particle suspension according to claim 1, in the determination of an analyte in a sample.

Inventors: NILSSON KURT G I, NILSSON, KURT, G., I.
Applicants: NILSSON KURT G I, PROCUR AB, NILSSON, KURT, G., I.
Application year: 1993
Main classification: G01N33
IPC codes: G01N33