SE431257B - SET AND DEVICE FOR IMMUNAL ANALYSIS WITH MAGNETIC METAL OR METAL OXIDE AS A BRAND - Google Patents

Description

The present invention relates to a method for determining the presence of selected immunochemical responses such as antibodies or antigens in body fluids. These selected substances are hereinafter referred to as analytical substances. g The determination of such analytical substances provides very useful medical information. This is done e.g. pregnancy tests, syphilis tests and blood factor tests are all now using conventional immunochemical methods. Most of these methods involve visual inspection of the formation of precipitated or agglutinated either-antibody complexes. These procedures sometimes require many repetitions at different reagent conditions to ensure the correct result and also require an individual, visual inspection for each test result.

Radioactive and fluorescent labeled substances have also been used to avoid subjective, visual determination. However, these processes are sometimes associated with problems such as the use of expensive or unstable branded substances or the sensitivity or safety of the handling of radioactive materials. In the method of the present invention, immunoreagents labeled with particles such as the presence of the assay substance in the body fluid are used. Similarly, in an indirect test, the immune reagent is reacted with the receptor-reagent but not with the receptor-reagent assay substance complex.

If no reactants are detected on the surface, the presence of the test substance in the body fluid is detected. In a competitive test, the body fluid and the immune reagent are placed on the surface at the same time.

The amount of reactants on the surface is a measure of the amount of analyte in the body fluid. This system can be used manually, but it can be easily adapted for automatic handling.

The invention is described in more detail with reference to the accompanying drawings, of which Fig. 1 describes a test card which is useful according to the invention, Fig. 2 is a schematic representation of the reactions included in the invention.

Known methods can be used to determine the presence of the reactants on the test surface. If the reactants e.g. affects the dielectric properties of the surface, a capacitance measurement reveals if the particles are present. In another example, if the particles are magnetic, magnetic sensing heads can be used such as those found in conventional tape recording equipment. Capacitance, conductivity and magnetic permeability measurements, which do not require direct contact with the test surface, are preferred, but measurements using direct contact between the surface and the detector are not excluded. In order to be able to detect the presence of the particles, it is preferred that the test surface is inert in relation to the property to be measured. However, it is possible to arrange the receptor reagent in bands on the surface or to induce a periodic magnetization on the surface such as that generated by a magnetic tape recording system and then pass the surface at a particular speed past a detector which registers the frequency. The determined frequency is a function of the placement of the bands of receptor reagents or induced periodic magnetization and the speed of movement of the surface.

Thus, the test surface itself need not be completely free of electrical reactance to be considered inert for the purposes of the invention. In a preferred system for determining the presence of a magnetic tag substance, the test surface is coated near a reference surface with magnetic particles. During the immune reactions, both the test surface and the reference surface can be magnetized in a periodic manner. The detector circuit can be made to determine only that part of the signal from the test surface which corresponds to the signal from the reference surface. This arrangement reduces the effects of system failure and provides increased sensitivity. The test surface must also be able to adhere to the receptor reagent and not interfere with the test result. The test surface may be smooth or have depressions or pits in which each test is performed. Although many materials are suitable test surfaces, plastic cards or strips are preferred. The test surface to which the receptor reagent is attached may be enclosed in plastic packages or bags. These packages may also contain the immune reagent in a separate compartment, which is intended to be added to the test surface after the addition of samples.

The presence of change in electrical reactance such as magnetic activity can be determined even when a very small amount of the reactant substance is present. Therefore, each test usually requires only one drop of body fluid, and the test surface can be very small. Consequently, a large number of separate test surfaces and, if desired, reference surfaces can be arranged on a single card or tape. The separate surfaces can be either a series of tests for different selected proteins or a series of tests for the same protein, when the concentration of the immune reagent on the test surface is varied within a range to show not only the presence of the selected protein but also its approximate concentration in body fluid. .

The receptor reagent can be attached to the test surface by surface absorption, gel entrapment, covalent bonding or other similar methods. Of these, covalent bonding is preferred. Any system for attaching the receptor so that the reagent molecules on the test surface are oriented so that they receive maximum activity is also preferred. The receptor reagent includes not only antibodies but also cellular receptor proteins or Tissue Receptor Proteins, Serum Transport Proteins and Lectins. Of these, antibodies probably have the most extensive applicability. Other classes of compounds such as chelating agents may also be valuable receptor reagents if they react with the rs1os51-2 substance to be determined in the body fluid, with sufficient specificity to avoid erroneous results caused by competing reactions.

The materials useful as reactant substances which combine with the immune compounds and form the immune reagent are those which alter the electrical reactance in the test surface.

This means that these materials, if distributed as a finely divided powder on the test surface, change the dielectric, conductive or magnetic properties of the surface. The advantage of the present invention consists in the use of particles, which can be detected in very small amounts of very sensitive but well-developed and easily accessible electrical components.

Furthermore, the use of such materials avoids the problems of transient activity and handling risks that exist when using radioactive labeled substances. The more preferred metals and metal oxides are those which exhibit ferromagnetism. Such materials can be reacted with the antibodies and applied to the test surface in a demagnetized state.

Once applied, the entire surface can be exposed to a magnetic field to magnetize the particles so that they can be detected.

This magnetic activity can then be easily detected with various well-known devices such as a conventional magnetic tape system or a Hall effect detector. Magnetic materials include, but are not limited to, metals and alloys such as iron powder, nickel, cobalt, CrO2, "Ferrofluid" (a ferromagnetic fluid, manufactured by Ferrofluids Corporation), C00, NiO, Mn2O3, magnetic plumbit compounds, magnetic iron oxides and “Alnico”, an alloy of aluminum, nickel, cobalt and copper, other valuable materials are organic charged complexes with high electrical conductivity such as N-methylphenazine tetracyanokinodimethane, [Öe (NO3) 6Mg (Hí) 6] 3.6 H2O crystals, Bi2Se3 crystals and tetrahiofulvalene complexes with tetracyano-β-quinodimethane or K2Pt (CN4) BrO3.H2O and amorphous materials with magnetic properties such as the chalcogenides, eg europium chalcogenides and chalcogenic glass particles.

Preferred magnetic materials are the magnetic oxides of iron, cobalt, nickel, chromium and manganese and oxide coated particles of iron or nickel. : f-s fi .oss 1-2 To provide a sufficient surface area for attaching the immune compounds, the reactants should preferably have a large surface area, at least 100 m2 / g. Particles with a diameter in the range 0.01-50 .mu.m are preferred.

These materials can be attached to the immune compounds by known methods. The reactants can be encapsulated with an organic polymer to which the immune compounds can be bound, or the surface can be silanized by conventional methods. Organic compounds can then be attached to the silane bond. U.S. Pat. No. 3,954,666 discloses polymer encapsulations of core materials, and U.S. Pat. No. 3,983,299 discloses the use of silane bonds to bind organic compounds to inorganic particles. Methods for making enzymes immobile on magnetic carriers, which methods could just as well be applied to antibodies, are described in methods in Enzymology, 44 p. 324-326. The manner in which the reactive organic compounds are attached to the reactants is not the subject of the present invention. Thus, other binding methods can also be used as long as they do not interfere with the immunocompounds of the immune compounds; hmmm * the compounds are selected so that they are specifically reactive with either the receptor reagent or with the complex between the receptor reagent and the substance in the body fluid to be determined.

Since the method of the present invention utilizes electronic and not visual reading, it can be easily adapted to automatic methods. The term "automatic" does not mean excluding the possibility that some of the procedures may be performed manually.

Fig. 1 shows a preferred test card 1 provided with several surfaces 2, to which receptor reagents are bound. Separate surfaces with different receptor reagents may be provided, or the same receptor reagent may be bound to each surface in different concentrations.

The card has a printed test identification 3 and a code 4, which can be read by a machine, and which contains the necessary information for automatic handling. Optionally, the card may have a space 5, where a patient's identification code can be entered.

Fig. 2 schematically shows a test surface 2 on a test card in Fig. 1. The receptor reagent 10 binds to the test surface. Body fluid from a patient, containing the antibodies II, the presence of which is to be determined, is added to each test surface. The antibody II, if present, forms a complex 12 with the receptor reagent. The test surface is then exposed to the immune reagent 13, which consists of an immune compound 14, bound to a reactant substance 15. This immune reagent forms complex 16 with the complex between the receptor reagent and the patient's antibody, if any, on the test surface.

A preferred system for carrying out the invention consists of a station in which the required test cards for the desired tests are inserted. Cards can be inserted individually or in groups as needed. The cards are automatically passed to a test additive station, where a drop of a patient's body fluid is applied to each test surface of the card. An automatic device may include a temperature control and a station for balancing the body fluid on the test surfaces for a predetermined time. The card is then passed to a station, where the immune reagent is applied to each test surface. Again, there may be systems for temperature control and equilibrium setting. Thereafter, excess immune reagent is removed, and the card is then examined for the presence of reactants remaining in the test by measuring changes in electrical reactance at the test surfaces of the card.

It should be noted that in the special described. In this embodiment, the test card is passed through each station. Other embodiments where each operation is performed on a card that remains at a single location in an automated or semi-automated device may also be used.

The method of this invention can be used in the determination of many different materials including, but not limited to, antigens and antibodies of viral, bacterial, cellular and human origin in addition to hormones, drug metabolites and specific proteins. The procedure is e.g. applicable in the determination of human antibodies, e.g. anti-thyroglobulin, antigenic proteins such as complement factors, protein hormones, immunoglobulins and free light and heavy chains, e.g. thyroxine-binding globulin and various types of micro-organism antigens and micro-organism antibodies such as hepatitis A and B viruses. The use of the inventive concept is primarily dependent on the availability of suitable material, which can serve as a receptor reagent, specifically reactive with the substance to be determined. a protein buffer solution containing 0.127% bovine serum albumin and phosphate buffered saline at pH 7.7 in 0.2. Sodium azide 0.1%, an antimicrobial agent, can be added to the diluted sample as a preservative.

The diluted patient sample can be quantitatively transferred to a test reaction card. The solid phase or carrier consisting of anti-human IgG, covalently bound to derivatized polyamide sheets, is prepared as follows: Nylon-6 film (10 g) can be suspended in BN HO 1 (300 ml) and stirred for four hours at 30 ° C, is removed from the solution and washed extensively with water, ethanol and ether. The carboxyl content of a nylon-6 film, which is hydrolyzed for four hours, is about 30-90 μmol / g. The coupling of the antibody to the depolymerized nylon-6 support can be performed by carbodiimide activation.

The overall reaction involves the condensation between the carboxyl group on the support and the amine group on the antibody. The reaction is carried out in two steps.

One gram of the acid-treated nylon-6 film is added to a solution of 50 mg of 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide-metho-p-toluenesulfate in 10 ml of water. The reaction mixture is adjusted to pH 4-5 with 6N H01, and the reaction is allowed to proceed at 30 ° C for 2-Ä hours. The film is then removed and washed several times with water to remove excess carbodiimide.

The activated film is then introduced into a solution containing commercially available anti-human IgG in concentrations of 1-50 mg of protein in 10 ml of water. After adjusting the solution to a pH between H and 5, the reaction is continued for 2-4 hours at room temperature or overnight at ° ° C. The reaction solution is then decanted, and the film is washed with water or buffer.

The test surface can then be equilibrated with the diluted sample for a sufficient time for antibody binding to occur. The first equilibrium can typically occur within 30 minutes. at 52 ° C. The reaction zones can then be washed with the same protein buffer solution and shaken to wash away unabsorbed antigen from the surface.

The test reagent zones can then be immersed in an excess of ge / tanti-human IgG, labeled with magnetic particles.

In order for equilibrium to enter the card, the immune particles are suitably diluted in phosphate buffer-physiological saline at pH 7.5 in 0.5 containing 0.12% bovine serum albumin. After equilibration, the test reaction surfaces can be washed with BSA phosphate buffer iv PH 7.7 in 0.2- The reaction surfaces are magnetized in a magnetic field, and the test card is passed through a detector, which can determine the presence of magnetic particles.

Example 2 This example shows the determination of antibodies directed against specific antigens.

A serum sample was diluted with protein buffer solution containing 0.1% bovine serum albumin and tris- (hydroxymethyl) -aminomethane (Tris) buffered at pH 6.5 ¿0, ß. Sodium azide, 0.1%, an antimicrobial agent, was added to the diluted sample as a preservative.

Dauw fi mawwæöw fi üæsmm fi m fi wt fl lm test reaction surface. The solid phase, the carrier, consisting of human serum albumin, covalently bonded to derivatized polyamide or polymethacrylic acid polymers was prepared as follows: The total coupling reaction between albumin and films may involve crosslinking between the amino or carboxyl groups of the film and the protein. Crosslinking can be accomplished using a polyfunctional aziridine reagent that is highly reactive with materials containing active hydrogens.

The films for binding human serum albumin were first suspended in a 1% solution of XAMA polyfunctional aziridine (Cordova Chemical, Sacramento, California). After stirring at 27 ° C for 30 minutes, the films were removed from the solution and washed with distilled water. The washed films were then stirred in a solution containing human serum albumin (1 mg / ml) for 12 hours at H00. The immobilized albumin film reagent can then be washed repeatedly with * snow buffer (pH 6.5 3 0.11) at 14 ° C. The weighting is continued until the albumin is no longer released from the film.

Representative levels of albumin binding to the films are given below: Film (ug / ug) Surfaces Surlyn Ga 3.5 Mylar <) ionomeric resin polyester (flame / treated) 7.3 no surface groups e polycarbonate nylon (HCl treated) 16.0 activated surface groups ethylene-maleic anhydride 31.0 The test surface for anti-HSA was equilibrated with the diluted sample for a sufficient time for anti-HSA binding to take place. The reaction surface was then washed with the same Tris buffer solution (pH 7.4 l 0.4), used in Example 1, to remove unbound sample residues from the surface.

The test reagent card was then immersed in an excess of goat anti-human serum albumin labeled with magnetic particles.

For equilibrium, the immune anti-HSA particles were appropriately diluted in Tris buffer at pH 6.5 3 0.5 containing 0.12% goat serum albumin. After equilibrium, the test reaction surfaces were washed free of unbound particles with goat serum albumin-Tris buffer.

The magnetic particles remaining on the reaction surface can be magnetized in response to a magnetic signal of known frequency. The test card can then be passed through a detector, which can detect the presence of the magnetic particles.

The detection of the reactance properties of the test surface can be facilitated by instrument procedures according to the phase-locking principle, with phase-sensitive rectification and other common electronic amplifier systems known to those skilled in the art.

The terms "substantially consisting of" and "substantially characterized by" do not exclude further process steps or components which do not prevent the benefits of the invention from being realized.

L_

Claims (5)

w oo ... i o m 01 -n-à I h) li Patentkrav A method of determining the presence of an assay substance in a body fluid, wherein a) the body fluid is contacted with a surface coated with receptor reagent that can specifically react with the assay substance, b) the coated surface is contacted with immune reagents, containing immune compound which can specifically react with the receptor reagent or with a complex of the assay substance and the receptor reagent, which immune compound is combined with reactant substances which can affect electrical reactance, c) unreacted immune reagent is removed from the surface and d) the electrical reactance is measured, characterized in that the reactant substances are magnetic metals and metal oxides. 2. A method according to claim 1, characterized in that the reactants are particles selected from the group consisting of the magnetic oxides of iron, cobalt, chromium, nickel and manganese, as well as oxide-coated particles of iron and nickel. 3. A method according to claim 1, characterized in that the receptor reagent is bound to the surface by adsorption, gel entrapment or covalent bonding. 4. A method according to claim 1, characterized in that the surface is attached to test information, which can be read by machine. Automated assay system for determining the presence of an assay substance in a body fluid, wherein a) the body fluid is contacted with a surface coated with receptor reagent that can specifically react with the assay substance, b) the coated surface is contacted with immune reagents, containing an immune compound which can specifically react with the receptor reagent or with a complex of the assay substance and the receptor reagent, which immune compound is combined with reactant substances which can affect electrical reactance, A) unreacted immune reagent is removed from the surface and the electrical reactance of the surface is measured, which analysis system is characterized in that it includes a test surface with a receptor reagent attached thereto, which receptor reagent specifically reacts with a component of a body fluid, a station for applying a sample of a body fluid to the test surface, device for applying an immune reagent to the test surface, which immune reagent consists of immunophenine which react specifically with the receptor reagent or with a complex of the receptor reagent and the component of the body fluid, which immune compounds are coated on reactant substances, comprising particles which can affect electrical reactance, a device for removing excess immune reagents and a device for determining the reactants on the test surface.