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
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
• • 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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

• the present invention relates to a new type of enzyme protein conjugate to be used
• radioimmunoassay 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.
• EIA enzyme immunoassay
• ELISA enzyme linked im unosorbent assay
• EIA is in many cases limited by insufficient sensitivity.
• 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).
• 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.
• the particles can consist of a polymer substance which can consist of natural, semisynthetic or synthetic materials.
• 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.
• 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.
• hydrophobic for example polystyrene particles, which can be nitrated, reduced and diazotized
• 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.
• nucleophilic groups amines, thiols
• the enzyme and the protein is activated with for example periodate or carbodiimide, instead of activation of the particle.
• the enzyme can be bound first and thereafter the antibody or the protein.
• antibodies can be bound via their thiol groups or via a smaller fragment of the antibody.
• 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.
• 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.
• 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.
• 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).
• crosslinker e.g glutaraldehyde or maleiimide
• 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.
• a covalent enzyme conjugate for example a soluble monoconjugate between enzyme and protein
• a noncovalent conjugate between enzyme and protein for example PAP (peroxidase- antiperoxidase conjugate)
• 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.
• 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.
• 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.
• the activity of either the bound or non-bound fraction is determined.
• 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
• P is product, which is detected
• -O- is the particle and / is the solid phase:
• 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.
• peroxidase-antiperoxidase conjugate PAP- 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.
• 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.).
• 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):
• 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).
• 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).
• 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.
• A Peroxidase-latex-antibody conjugate prepared via periodate activation of peroxidase carbohydrate .
• Horseradish peroxidase (Boehringer; 16 mg; 250 ⁇ /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 .
• An amino group containing particle preparation (15 mg dry weight of particles; 0.1 ⁇ m 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.
• NaCNBH 3 (450 ⁇ l; 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.
• MOPS-HC1 3- (N-morpholino) propanesulfonic acid-HCl
• 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.
• 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.
• diazonium group containing particles are very reactive against hydrophobic groups on enzymes and proteins, antibodies (e.g. at pH 7-9) .
• enzyme and protein are added to the activated particles simultaneously or after each other.
• 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.
• 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.
• 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.
• Example 1A 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.
• Immunopure ⁇ activated peroxidase Pierce, Rockford, USA
• 1 ml cold, distilled water 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.
• Staphylococcus aureus was added instead of the rabbit antibody in 1.B.3.
• PAP a noncovalent, soluble conjugate between horseradish peroxidase and rabbit antibody to horseradish peroxidase; Dakopatts, Denmark; 1.2 mg; dissolved in 920 ⁇ l buffer E
• a latex particle suspension carrier protein
• the mixture was allowed to stand for 45 minutes at ambient temperature.
• a microtiter plate was coated with rabbit antibodies to human alfa-1-fetoprotein (immunoglobulins; Dakopatts, Denmark;
• the plate was washed with buffer A containing 0.1 % Tween 20 and
• buffer B 0.5 M NaCl
• 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.
• Substrate solution 100 ⁇ l 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 ⁇ l of 30% H2O2; hereinafter called buffer C
• buffer C citric acid-phosphate buffer, pH 5.0, and 12 ⁇ l of 30% H2O2
• a microtiter plate was coated overnight at 4 °C with human alfa-1-fetoprotein (200 ⁇ l; 2 ⁇ g per ml of buffer A) .
• 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.
• Swine antibodies to rabbit immunoglobulins (Dakopatts, Denmark) was added (0.5 mg and 100 ⁇ l in each well) and was incubated for 1 h at room temperature.
• Conjugate l.D (diluted 20 times with buffer B; 100 ⁇ l in each well) was incubated for 1 h at room temperature.
• 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) .

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