CH638052A5 - METHOD AND AUTOMATED ANALYSIS DEVICE FOR DETERMINING AN IMMUNOCHEMICALLY REACTIVE SUBSTANCE IN A BODY LIQUID. - Google Patents

Description

The invention relates to a method for determining the reagent is generally applied as a liquid suspension to the presence of immunochemically reactive substance surface. Unreacted immune reagent, such as antibodies or antigens, in the body is then removed from the surface and the surface on liquids, and an automated analysis device changes the dielectric constant, the conductivity to carry out the method. or magnetic permeability. With a direct

In the test, the immunoreagent is reactive with the receptor reagent, immunochemically reactive substance complex, and if it is determined that the reactance marker particles are bound to the surface, this indicates the presence of the immunochemically reactive substance in the body fluid. Similarly, in an indirect test, the immunoreagent is reactive with the receptor reagent, but not with the receptor reagent, immunochemically reactive substance complex. If no reactance marker particles are detected on the surface, this indicates the presence of the immunochemically reactive substance in the body fluid. In a competition test, the body fluid and the immune reagent are applied to the surface at the same time. The amount of reactance marker particles on the surface is a measure of the amount of immunochemically reactive substance in the body fluid. This system can be used manually, but can be easily adapted to automated working methods.

Embodiments of the invention are described below with reference to the figures.

Figure 1 is an illustration of a test card suitable for the purposes of the invention.

2 is a schematic representation of the reactions taking place within the scope of the invention.

Common means can be used to determine the presence of the reactance marker particles on the test area. For example, if the reactance marker particles affect the dielectric properties of the surface, a capacitance measurement indicates whether the particles are present. If, to give another example, the particles are magnetic, magnetic heads such as those found in conventional sound recorders can be used. Capacitive, conductive, and magnetic permeability measurements that do not require direct contact with the test surface are preferred, but measurements with direct contact between the surface and the detector are not excluded. In order to determine the presence of the particles, the test surface is preferably indifferent to the property to be measured. However, it is possible to strip the receptor reagent on the surface or induce periodic magnetization on the surface, as generated by a magnetic tape recording system, and then move the surface past a detector at a certain rate to monitor the frequency. The observed frequency is a function of the arrangement of the strips of the receptor reagent or the induced periodic magnetization and the speed of movement of the surface. The test surface itself therefore does not have to be completely free of electrical reactance in order to be regarded as indifferent for the purposes of the invention. In a preferred system for determining the presence of the magnetic marker particles, the test surface is adjacent to a reference surface covered with magnetic particles. After the immune reactions, both the test surface and the reference surface can be magnetized periodically. The detector circuit can be designed to receive only the portion of the signal from the test surface that is in phase with the signal from the reference surface. This arrangement reduces the effects of equipment noise and gives increased sensitivity.

The test surface must also be able to bind the receptor reagent and must not falsify the test result. The surface can be flat or have depressions in which each test is carried out. Numerous materials are suitable for the test surfaces, however, plastic638 052

fabric cards or ribbons preferred. The test surface to which the receptor reagent is bound can be in closed plastic packages or bags. These packages can also contain the immunoreagents in a separate area which is intended for application to the test surface after the addition of the sample.

A change in electrical reactance, for example magnetic activity, can be determined even if only a very small amount of the reactance marker particles is present. Therefore, each test usually requires only one drop of body fluid and the test surface can be very small. As a result, a large number of separate test areas and, if desired, reference areas can be arranged on a single card or tape. The separate areas can be used either for a series of tests for different selected proteins or a series of tests for the same protein, if the concentration of the immunoreagent on the test surface is changed over a certain range, to provide information not only about the presence of the selected one Protein, but also to maintain its approximate concentration in the body fluid.

The receptor reagent can be bound to the test surface by surface adsorption, gel confinement, covalent binding or by other similar methods. Of these, covalent bonding is preferred. Any system of receptor binding that can orient the reagent molecules on the test surface so that they have maximum activity is also preferred. Not only antibodies, but also tissues or cell receptor proteins, serum transport proteins and leptins (Ringertive and Savate “Cell Hybrids”, Academic Press 1976, pp. 55 and 247) are suitable as receptor reagents. Of these, the antibodies probably have the greatest application. Other classes of compounds, for example chelating agents, can also serve as useful receptor reagents,

if they react with the substance to be determined in the body fluid with sufficient specificity to avoid false results caused by competitive reactions.

As materials which are suitable as reactance marking particles which combine with immune compounds to form the immunoreagents, compounds which alter the electrical reactance of the test surface, i.e. these materials, when distributed as a fine powder on the test surface, change the dielectric, conductive or magnetic properties of the surface. The advantage of the invention is that particles are used which can be detected and detected in very small amounts by very sensitive, but well-developed and easily available electrical devices. Furthermore, the use of these materials eliminates the problems of inconsistent activity and the handling risks that arise when using radioactive indicators. Metals and metal oxides are preferred as reactance markers. Metals and metal oxides which have ferromagnetism are particularly preferred. These materials can be reacted with the antibodies and applied to the test surface in the demagnetized state. After application, the entire surface can be exposed to a magnetic field to magnetize the particles for identification and detection purposes. This magnetic activity can then easily be determined and detected using various known means, for example a conventional magnetic tape system or a Hall effect detector. Examples of suitable magnetic materials are metals and alloys such as iron powder, nickel, cobalt, Cr02, the ferromagnetic fluid “ferrofluid”

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(Manufacturer Ferrofluids Corp.) CoO, NiO, MmCh, Magneto-plumbite, magnetic iron oxides and the product «Alnico», an alloy of aluminum, nickel, cobalt and copper. Also suitable are organic charge complexes with high electrical conductivity, for example N-methylphenazinumtetracyanchinodimethane [Ce (N03) «Mg (H20) 6] 3. óHìO crystals, BÌ2Se3 crystals and tetrahiofulvalene complexes with tetrocyan-p-quinodimethane or K2Pt (CN <t) BrOa.HîO and amorphous materials with magnetic properties, e.g. the chalcogenides, e.g. the europium chalcogenide and chalcogenide glass particles.

The preferred magnetic materials are the magnetic oxides of iron, cobalt, nickel, chromium and manganese and particles of iron or nickel coated with oxide.

In order to obtain a sufficiently large surface area for binding the immune compounds, reactance markers with a large surface area of at least 100 m2 / g are preferred. Particles with a size in the range from 0.01 to 50 μm are particularly preferred. These materials can be bound to immune compounds by known methods. The reactance markers can be embedded in an organic polymer to which immune compounds can be bound, or the surface can be coated with silane-containing substances in the customary manner, and organic compounds can then be bound to the silane bond, US Pat. No. 3,954,666 describes the embedding of core materials in polymers, and US Pat. No. 3,983,299 describes the use of silane bonds to form organic compounds Methods for immobilizing enzymes on magnetic carriers, which would also be applicable to antibodies, are described under “Methods in Enzymology” XLIV, pages 324-326, The type of binding of the reactive organic compounds the reactance markers do not form the basis ndlage of the invention. Other binding methods can also be used as long as they do not impair the ability of the immune compounds to form complexes. Three immune compounds are chosen in order to be specifically reactive either with the receptor reagents or with the complex of the receptor reagent with the substance to be determined in the body fluid.

Since the examination or verification in the methods according to the invention is carried out electronically and not visually, the method can easily be adapted to automated designs. The term "automated" used here is not intended to exclude the possibility that some of the operations can be carried out manually. 1 shows a preferred test card 1 with a plurality of areas or fields 2, to which receptor reagents are bound. Separate fields with different receptor reagents can be provided, or the same receptor reagent can be bound to each field in different concentrations. The card has a printed test name 3 and a machine-readable code 4, which contains the necessary information for automated work. The card can optionally have a space 5 for entering a patient identification code.

FIG. 2 schematically illustrates a test field 2 of the test card shown in FIG. 1. Receptor reagents 10 are bound to the test field. Body fluid of the patient, which contains the antibodies 11, the presence of which is to be determined, is applied to each test field. The antibody 11, if present, forms a complex 12 with the receptor reagent. The test field is then exposed to an immune reagent 13, which consists of an immune compound 14, which is bound to a reactance marker 15. This immunoreagent forms complex 16 with the receptor reagent-patient-antibody complex, if it is present, on the test field.

A preferred device for carrying out the invention would include a station for entering the necessary test cards for the desired tests. The cards can be entered individually or in groups as required. The cards are automatically routed to a sample addition station where a drop of a patient's body fluid is applied to each test field on the card. An automated device may include temperature control and a body fluid equilibration station on the test fields for predetermined times. The card is then taken to a station where the immunoreagent is applied to each test field. This station can also include temperature control and equilibration systems. Excess immune reagent is then removed and the card is then checked for the presence of reactant markers that have remained in the test by measuring changes in the electrical reactance of the test fields on the card.

It should be noted that the test card in the particular embodiment described is passed through each station. Other embodiments in which each operation is performed on a card held in a single location in an automated or semi-automated device are also possible.

The method according to the invention is applicable to the determination of the most diverse materials including the antigens and antibodies, the origin of which is from viruses, bacteria, cells and humans, as well as to hormones, metabolites of medicaments and special proteins. For example, the method is suitable for the determination of human autoantibodies, e.g. antithyroglobulin, antigenic proteins such as complement factors, protein hormones, immunogobulins and free ligth and heavy chains, e.g. Thyroxinbundungsgobu-lin, and various types of microorganism antigens and antibodies, such as hepatitis A and B virus. The application of this method depends primarily on the availability of a suitable material that serves as a receptor reagent and is specifically reactive with the substance to be determined.

example 1

To determine an IgG human antibody, a serum sample can be prediluted with a protein buffer solution containing 0.12% bovine serum albumin and phosphate-buffered saline at pH 7.7 ± 0.2. 0.1% sodium azide, an antimicrobial, can be added to the diluent as a preservative.

The diluted patient sample can be transferred quantitatively to a test reaction card. The solid phase support, which consists of anti-human IgG, which is covalently bound to derivatized polyamide films, is produced as follows: 10 g of film made of nylon-6 is suspended in 300 ml of 3n-HC1 and 4 hours at 30 ° C stirred, removed from the solution and washed exhaustively with water, ethanol and ether. The carboxyl content of a 4 hour hydrolyzed nylon-6 film is about 30 to 90 (iMol / g.

The coupling of the antibody to the depolymerized nylon-6 support can be carried out by activation with carbodiimide. The overall reaction consists of the condensation between the carboxyl group on the support and the amine groups of the antibody. The reaction is carried out in two stages.

1 g of the acid-treated nylon 6 film is added to a solution of 50 mg of l-cyclohexyl-3- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfate in 10 ml of water. The

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The reaction mixture is adjusted to pH 4 to 5 with 6N HCl and left to react at 30 ° C. for 2 to 4 hours. The film is then removed and washed several times with water to remove the excess carbodiimide.

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

The test surface is then equilibrated with the diluted sample for a sufficient time to allow antibody binding to take place. The first equilibration can generally take about 30 minutes at 32 ° C. The reaction zones can then be washed with the same protein buffer solution and shaken to rinse unabsorbed antigen from the surface.

The test reagent zones can then be immersed in excess goat anti-human IgG labeled with magnetic particles. For equilibration on the card, the immune particles are appropriately diluted in phosphate saline buffer containing 0.12% bovine serum albumin at pH 7.5 ± 0.5. After equilibration, the test reaction areas can be washed with the BSA phosphate buffer at pH 7.7 ± 0.2.

The reaction areas are magnetized in a magnetic field, whereupon the test card is passed through a detector which is able to determine the presence of magnetic particles.

Example 2

This example illustrates the determination of antibodies directed against specific antigens.

A serum sample was prediluted with a protein buffer solution containing 0.1% bovine serum albumin and tris (hydroxymethyl) amino methane (Tris) and buffered at pH 6.5 ± 0.4. 0.1% sodium azide, a micro-killing agent, was added to the diluent as a preservative.

The diluted sample was transferred quantitatively to a test reaction area. The solid phase support, which consisted of human serum albumin covalently bound to either derivatized polyamide or polymethacrylic acid polymers, was prepared as follows:

The overall coupling reaction between albumin and films can be linked to a cross-linking between the amino groups 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 hydrogen atoms.

Films for binding human serum albumin

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were first suspended in 1% solution of polyfunctional Aziri-din XAMA (Cordova Chemical, Sacramento, California), the suspension was stirred at 27 ° C. for 30 minutes, after which the films were removed from the solution and washed with distilled water. The washed sheets were then stirred in a solution containing human serum albumin (1 mg / ml) for 12 hours at 4 ° C. The immobilized albumin and foil reagent is then washed repeatedly with Tris buffer (pH 6.5 ± 0.4) at 4 ° C. Washing continues until no more albumin is released from the film. Representative concentrations at which the albumin is bound to the films are listed below.

Foil p.g / cm2

Surface groups Surlyn 3.3

"Mylar" ionomer resin

Polyester (flame treated) 7.3

No surface groups polycarbonate

Nylon (HCl treated) 16.0

Activated surface groups ethylene maleic anhydride <1.0

The test surface for anti-HSA was diluted with the diluted sample so long that the anti-HSA binding could take place. The reaction surface was then washed with the same Tris buffer solution (pH 7.4 ± 0.4) used in the experiment described in Example 1 to remove unbound sample residues from the surface.

The test reagent card was then immersed in excess goat anti-human serum albumin labeled with magnetic particles. For equilibration, the immune anti-HSA particles were appropriately diluted in a Tris buffer (pH 6.5 ± 0.5) containing 0.12% goat serum albumin. After equilibration, the test reaction areas were washed with the goat serum albumin-Tris buffer to remove unbound particles.

The magnetic particles remaining on the reaction surface can be magnetized by a magnetic signal of known frequency. The test card can then be processed in a detector that can detect the presence of magnetic particles. The determination of the reactance properties of the test area can be done by instrument techniques, e.g. the technique of phase lock application, phase sensitive rectification and other common electronic amplification systems known to those skilled in the art are facilitated.

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1 sheet of drawings

Claims (9)

638 052 1. A method for determining the presence of a punch allows many valuable medical statements. In the case of an immunochemically reactive substance in a body fluid, for example, the pregnancy test, the syphilis test, are characterized in that all and the blood factor test are carried out according to the usual immunochemical a) a surface covered with a receptor reagent 5 see methods performed. Most of these metho-ist, which are specific to the immunochemically reactive substance, are visually checked for the formation of fishes that are reactive with the immunochemically reactive or agglutinated antigen-antibody complexes. liquid containing substance in these methods sometimes requires multiple repetitions of contact with different ratios of reagents to ensure accurate b) to ensure the covered surface with an immunoreagent in io keit, as well as an individual visual verification form of an immune connection in contact with each test result. Furthermore, radioactive and fluid receptor reagents or with a complex of the immunorescent labeling substances are used in order to avoid specific determination of the subjective chemically reactive substance and the receptor reagent. These methods throw fish is reactive and sometimes have problems with reactant markers, either in terms of use, which are in the form of particles that can affect the elec- tricity of expensive or unstable markers, or chemical reactivity sensitivity or safety and handling c) unreacted immune reagent from the surface of radioactive materials. According to the invention, immune reagents are removed which use particles, for example d), to measure the electrical reactance of the surface. magnetic materials that are safe to use 2. The method according to claim 1, characterized in that 20 and easily with highly sensitive and easily available elek- 'that the reactance markers magnetically sensitive tronic devices are detectable, are marked. These are. Method can easily be automated and semi-automated 2nd PATENT CLAIMS The determination of an immunochemically reactive substance 3. The method according to claim 1, characterized in that the method of operation can be adapted. that the reactance markers have a capacitive reactance. The use of magnetic particles in the sero. 25 logical tests are known, but the magnetic 4. The method according to claim 1, characterized, see particles before the invention only for removal or that the reactance markers are conductive. Complex binding of various components from a river 5. The method according to any one of claims 1 to 4, thereby used liquid sample. For example, the US marks describe that the reactance markers in US Pat. Nos. 4018886 and 3970518 consist of the use of magnetically significant metal and / or metal oxide particles. 30 active particles for the isolation of selected proteins with 6. The method according to claim 5, characterized in that subsequent cleavage of the proteins from the magnetic part - that the particles consist of magnetic oxides of iron, cooking and a visual check for precipitated proteins, balt, chromium, nickel or manganese or of oxide U.S. Patent 3,933,997 describes the use of magnetically clad particles made of iron or nickel. see particles to concentrate radioactive markers 7. The method according to claim 1, characterized in 35 substances on a test substance. that the receptor reagent by adsorption, Geleinschlies- None of the publications mentioned describes the solution or covalent bond to the surface using the magnetic determination to achieve the. desired result. 8. The method according to claim 1, characterized in that the method according to the invention for determining that the surface is additionally covered with machine-readable test in the presence of an immunochemically reactive substance in egg formations. ner body fluid is in the preceding claim 1 9. Automated analyzer marked for implementation. of the method according to claim 1, characterized in that the presence or absence of an immunoche- that it has the following parts: mixed reactive substance, for example a specific test surface to which a receptor reagent is bound, 45 antigen or antibody, in a body fluid this can be determined with an immunochemically reactive substance of a body, ie by taking a sample of the Body fluid-per fluid is specifically reactive, is applied to a surface with a Means for applying a sample of a body fluid to receptor reagent covered with the immunochemically reactive sub-die test surface, specifically reactive. If the Means for applying an immune response to the test surface, so if the immunochemically reactive substance is present, forms the surface, the immune reagent forming a complex with the receptor reagent. The surface holding that with the receptor reagent or with a complex is then combined with an immunoreagent that contains the receptor reagent with the immunochemically reactive sub- an immune component that is either specifically reactive with the receptor of the body fluid and on tor reagent or with a complex of the receptor reagent and reactance markers are applied which have the form 55 of the immunochemically reactive substance "specifically reactive-of particles which can be influenced by the electrical reactance. The immunoreagent further contains particles which are capable of doing so electrical reactance to be Able to remove excess immune reagent and influence. They are tiny fragments that detect the di-agents that determine the presence of reactance markers, constant electricity, conductivity, or magnetic per on the test surface. 60 Change surface meability. These particles are referred to as reactance marker particles in this case. Metal or metal oxide particles are preferred and magnetically active particles are particularly preferred. The immune