DE19808930A1 - Method for the detection of antigens or antibodies - Google Patents

Description

Method for detecting an antigen or antibody in a biological Liquid such as saliva, blood, lymph, faeces or urine by applying one Aggregation reaction method based on an antigen-antibody reaction rests.

So far, the determination of the proportion of a specific antibody in one Test medium such as blood and body fluids is an aggregation reaction procedure applied in the composite particles such as gelatin, kaolin or a synthetic polymer with the immobilized antigen for aggregation were. In this aggregation reaction process, the recording and assessment division with a test plate with several vessels carried out for serial Dilution of a test medium can be used. If at the acquisition If the aggregation reaction occurs (positive reaction), a mattenar becomes aggregation pattern on the walls of the vessels as an evenly distributed Ag gregat generated, while in the absence of an aggregation reaction (negative Reacti on) a circular, button-like precipitate is deposited on the bottom of the vessels is stored because it flows down from the walls.  

In the previously known tests of hemagglutination inhibition, however, it was him required to continue a cumbersome surgery to remove an inhibitor to provide, d. H. substances that inhibit an antigen-antibody reaction, such as lipid or glycoprotein in the serum. Furthermore, such a process requires inhibitor removal generally takes 24 hours.

The object of the invention is to provide a method for detecting an anti gene or antibody in a biological fluid to indicate that easier detection has a high degree of accuracy without an inhibitor need remote operation.

To achieve this object, the invention provides a method for detecting a Antigen or antibody in a biological fluid to be tested, the the following steps:

Applying the biological fluid to a test plate for the aggregation test, a binder for the antigen or the antibody adheres to the surface thereof, to add the biological fluid to each of the test plate vessels; preferably removing the biological fluid from the test plate and Rinse the test plate after the biological fluid for a predetermined Time was kept in the vessels; and entering a particle of the immobili based antigen or antibody-containing liquid into the vessels.

The invention is based on the finding that the above object can be solved by placing a test medium in each vessel of the test plate Aggregation test is entered, on the surface of which a binder for a Antigen or an antibody adheres, causing the antigen or the antibody is bound in the test medium and the binder, after which the rest Test medium is removed from each vessel of the test plate and the test plate for Remove a residue of the adhering inhibitor and other substances will.

When using this detection method for the antigen or antibody it becomes possible to operate on removing an inhibitor from the er Verification process using the aggregation reaction process zichten to avoid the formation of a pseudo-aggregation picture z. B. in the dilution of the test medium like a serum with low dilution  degree is considered, and the recording and assessment with high accuracy degree of ease.

The test plate for an aggregation test in the method according to the invention is not subject to any restrictions as long as they have multiple vessels to form one serial dilution of a test medium. In general, the test plate can be a Plate or strip with vessels, the bottom of each vessel a V-shaped, U-shaped or similar or modified cross section Has. The test plate can preferably consist of a plastic. Typical Examples are polystyrene, polypropylene and polyvinyl chloride as well as others.

In the invention, the test plate is applied after a binder for an antigen or an antibody applied to the bottom of each vessel was brought, d. H. on the inner surface of the V-shaped, U-shaped or the like Cross-sectional part of each vessel. For example, the test plate can be used after the inside of each tube with protein A and / or protein G or with an avidin, streptavidin or their derivatives was seen and the avidin, streptavidin or their derivative with a biotin labeling antigen or antibody has been reacted. Suitable Examples of the avidine derivatives are NeutraAvidin (available from Pierce Inc.) and UltraAvidin (available from Leinco Technologies Inc.).

In practicing the invention, a biological one to be tested Liquid, d. H. the test medium for the aggregation test in each tube entered the test plate. The test plate used has several tubes Recording the test medium and contains a binder for the antigen or Antibodies stick to a surface. In each tube there is one in the test tube dium containing antigen or antibody bound to the binder. Around To achieve a satisfactory binding effect, the test plate is preferred standing with the entered test medium for a predetermined time leave or shaken. The standing or shaking time is generally in the range of about 15 minutes to an hour, but is preferably at least 20 Minutes. Preferably, the test plate is shaken to make an effective connection between the antigen or antibody and the binding agent.

In previously known methods, after entering the test medium particles on the test plate and when holding the test medium in each vessel  of the immobilized antigen or antibody to be detected (generated by Immobilize on a particulate carrier) in the test plate vessels to generate an antigen-antibody response. However, this will inhibited in this method because an inhibitor contained in the test medium substance such as lipid or glycoprotein the particulate immobilized antigen or can cover the antibody.

In contrast, such inhibition of the antigen-antibody response can be avoided in the invention, since a test medium in entered each vial of the test plate and pre-entered the test medium is preferably removed from the test plate after it has been for a predetermined Time was held after which the test plate was rinsed to remove all of that Antibodies or antigen bound to the binding agent, various substances to remove zen. The test plate can be rinsed by rinsing the tubes gene, preferably the empty vessels after removal of the test medium, for what Water, a physiological saline or a physiological saline solution is used. After rinsing, the remaining inhibitor can be removed from the test plate, whereby the subsequent removal of the Inhibitor can be omitted, that as a special part of the previously known Procedure is necessary.

After rinsing the test plate, the immobilized antigen or the anti body into the tubes of the test plate. If a trace portion of the an antigen to be detected on a wall of the test medium remaining or contains antibodies can cause an antigen-antibody reaction with good accuracy and an antibody or antigen can be included in the test medium can be detected with high accuracy. Mat-like aggregation pictures are generated when any antibody or antigen is present in the test medium hold is.

The ver when detecting an antigen or antibody according to the invention immobilized antigen or the antibody is not subject ner special limitation. Preferably the immobilized antigen or the antibody is used as aggregating composite particles, that contain an antigen or an antibody that is on a composite body made of ceramic and polymer, latex, gelatin or kaolin. The Ag gregieren of composite particles made of ceramic and polymer with existing  immobilized antigens or antibodies is, for example, in the Japani United States Patent Application 7-174762 and U.S. Patent 5,540,995 wrote. These particles are by adsorbing and immobilizing an anti gene or antibody on a particulate composite body made of polymer particles with a layer of a calcium phosphate compound, wherein the polymer particles or the entire composite body colored and then not adsorbed sites of the composite body was treated with a blocking agent the. Preferably used as particulate composite bodies are by physically hitting the particles of the calcium phosphate compound can be made against polymer particles, creating the surface of the polymer particle is coated with the calcium phosphate compound.

A large number of calcium phosphate compounds can be used as long as they have a CNP ratio in the range of 1.0 to 2.0. For example, one member or a combination of two or more members of the group Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , Ca ₁₀ (PO ₄ ) ₆ F ₂ , Ca ₁₀ (PO ₄ ) ₆ C ₁₂ , Ca ₃ (Po ₄ ) ₂ , Ca ₂ P ₂ O ₇ , Ca ₄ O (PO ₄ ) ₂ , CaHPO ₄ can be used as calcium phosphate compound. Among these calcium phosphate compounds, hydroxylapatite and tricalcium phosphate are preferably used, a calcium phosphate compound containing hydroxylapatite as the main component is most favorable. When fluorapatite is used as the calcium phosphate compound, the proportion of fluorine in all calcium phosphate compounds should preferably not exceed about 5 percent by weight, since a fluorine proportion above about 5 percent by weight can cause fluorine to be released from such compounds. These calcium phosphate compounds can be produced by any known production method including wet synthesis, dry synthesis and others.

The particles of the calcium phosphate compound can be, for example, by Spray drying a slurry of the calcium phosphate compound the one after which the calcium phosphate particles are burned. Alternatively, too Any other granulation process in addition to this manufacturing process be used to create similar calcium phosphate particles. Preferential A screening process or other separation process can be used as an option Weeding out the calcium phosphate compound particles of a predetermined one Size range depending on the intended use of the particles the.

Examples

The invention is explained in more detail below with the aid of examples. she is but not limited to these. All proportions and percentages in the examples refer to weight unless otherwise stated.

Preparation example 1

Manufacture of ceramic and polymer composite particles with immobilized yes panic encephalitis virus of the Beijing strain.

50 g nylon particles with an average particle diameter of about 5 microns and colored with an anthraquinone color, commercially available under the name "Mitsui ML Colors ML red VF-2" from Mitsui Toatsu Senryo Co., and 7.5 g Hy Hydroxyapatite particles were in the Nara at 32 ° C to 50 ° C for about 5 minutes Hybridizing system NHS-1 mixed, which was rotated at 8000 U / m to the Coating the surface of the nylon particles with hydroxyapatite. With the so get Composite particles, the hydroxyapatite layer had an average thickness of 0.44 µm and an average particle diameter of 5.8 µm.

32 µg / ml of the Beijing strain Japanese encephalitis virus and one 10% (GN) solution of the composite particles were in an equivalent ratio mixed and kneaded in a rotator for about an hour to form an adsorp tion of the virus antigen on the composite particles. The composite Particles were centrifuged at 1000 rpm for about 5 minutes to be eliminated while the supernatant generated at the same time has been removed. The Granules were loosened and the antigen was added to the granules at 0.05% Glutaraldehyde as a dilute solution of phosphoric acid buffer solution with egg immobilized in a pH of 7.2 (hereinafter referred to as PBS). After Stirring at room temperature in a rotator, the particles with the im mobilized antigen centrifuged at 1000 rpm for approximately 5 minutes to grit Chen excrete while the supernatant generated at the same time removed has been. With a 1% (GN) solution of the "Block Ace" from Dainippon Seiyaku Co. masking agent was available for about an hour masking was performed to avoid areas not covered with adsorbed antigen masking the granules while stirring them in a rotator. After stirring in a rotator at room temperature, the particles were approx. 5 Mi  centrifuged at 1000 RPM for a period of time to excrete granules during the the supernatant generated at the same time was removed. To detach the Block Ace To prevent the granules from being diluted with 0.05% glutaraldehyde Solution of PBS (pH = 7.2), immobilized. After stirring at room temperature The granules were centrifuged in a rotator for about 5 minutes at 1000 rpm to excrete granules while the supernatant is being generated was removed. With 1% (w / v) solution of Block Ace became more active for inactivation Residues of the glutaraldehyde are masked, after which the particles are in a 0.4% (w / v) solution solution from Block Ace.

Preparation example 2 Manufacture of the agglomeration test plate

0.05 ml of the PBS solution with a protein A in a concentration of 1.0 × 10 ^-3 mg / ml was introduced into each tube of the polystyrene plate. The polystyrene plate used contained 96 V-shaped tubes with a capacity of 0.3 ml. After one minute, the PBS solution was removed from the polystyrene plate to remove the releasing protein A in the PBS solution in the tubes, and there were Pour 0.05 ml of the PBS solution into each empty vessel on the plate and then remove the PBS solution again. The plate was dried to obtain a test plate with the attached Protein A.

Preparation example 3 Manufacture of ceramic and polymer composite particles with immobilized Paragonimiasis antigen

The procedure of Preparation Example 1 was repeated to make the composite part Chen made of ceramic and polymer with immobilized Paragonimiasis antigen generate, provided that 25 µg / ml based on a portion of the Antigen protein from the paragonimiasis antigen instead of the Japanese en Beijing strain cephalitis virus was used.  

example 1

100 µl serum (test medium) of a patient with Japanese encephalitis, ver thinned with PBS to generate ten times the volume, were in each tube the first row of the test plate provided with protein A from the preparation case game 2 entered, and 50 ml of the PBS solution with 10% Block Ace were in each of the remaining rows of the test plate. Then the first row of the test plate, dilute with 50 µl PBS and one dilution generating factor of 2n, d. H. a double serial dilution. After about The test medium was shaken from all tubes for one hour removed, and the test plate was rinsed with water and then dried. There after 100 ml of the 0.1% solution of the ceramic and ceramic composite particles Polymer with Beijing immobilized Japanese encephalitis virus Strain from preparation example 1 into the empty containers of the dried Test plate entered. After about two hours it was observed that clear positive images could be generated from the serum dilution factor 10.

Example 2 1. Production of the plate for the determination of IgG especially for paragonimia sis

50 µl of the avidin solution, commercially available under the name "NeutraAvidin" from Pierce Co. with a carbonate buffer solution (pH = 9.6) for Er Witnesses of 25 µg / ml were placed in each tube of the Glynor microtitration plate Co. entered. The microtitration plate had 96 (12 × 8) V-shaped tubes. The Solution was removed from the plate after being left for about an hour Room temperature. In the avidin-immobilized thus obtained The plate was rinsed with deionized water and then with 100 µl a four times diluted solution of Block Ace (in PBS, 1% by weight per Solution). The plate was left to stand at room temperature for one hour and then the diluted Block Ace solution was removed from the plate distant. It was air dried to form an avidin immobilized plate receive.

After 0.5 mg / ml of the anti-human IgG biotin-labeling antibody four hundred times the volume was diluted, 50 ul of the ver  diluted solution is added to each vial of the avidin immobilized plate. The The plate was left to stand at room temperature for about an hour Binding reaction between the avidin and the biotin. It revealed a plate to determine the IgG especially for paragonimiasis.

2. Determine human serum

A human serum that is positive for the Paragonimiase and a human Se rum, which is negative for the Paragonimiase, were tenfold each Volume diluted. In the plate produced according to (1.) above, the ver thin serum was poured into the first twelve vessels and a ver generated thinning with a factor of 2n, d. H. a double serial dilution. In Each vial contained 50 µl of the diluted serum to be tested. Under Under the same conditions, the plate was kept at room temperature for about an hour left standing.

After that, the plate was rinsed with deionized water, followed by each jar with 100 µl of the 0.1% solution of the ceramic and polymer composite particles the immobilized paragonimiasis antigen from Comparative Example 3 has been. The plate was left standing and after an hour it was judged whether an aggregation picture was created or not. It was from the results too recognize that clear positive images in the dilution range from 10 to 20480 for the paragonimiasis positive serum and also clear negative images in the dilution Range from 10 to 20480 obtained for the paragonimiasis negative serum could become.

Comparative Example 1

100 ul of the serum (test medium, in accordance with that from Example 1) was into each tube in the first row of the test plate provided with protein A. entered the reference example 2, and 50 ul of the PBS solution with about 10% Block Ace were placed in each jar of the remaining rows of the test plate. Then the first row of the test plate was diluted with 50 µl PBS in each dilution with a dilution factor of 2n, i.e. H. a double Serial dilution was generated and the test plate was left for about an hour shaken. Then 50 ul of the 0.1% solution of the composite particles were Ceramic and polymer with the immobilized Japanese encephalitis virus des  Peking strain from reference example 1 into each vessel of the test plate given. After about two hours, the assessment showed that pseu do-negative reaction images in the serum dilution range from 10 to 80 could be witnessed, and that positive reaction pictures with increasing ver thinning factor over 80 could be generated.

Claims (4)

1. A method for detecting an antigen or antibody in a biological fluid to be tested, characterized by
Entering the biological fluid into at least one vessel of a test plate, on the surface of which a binding agent for the antigen or the antibody adheres,
Holding the liquid in the vessel for a predetermined time, and
Enter a liquid into the vessel that contains a particulate immobilized antigen or a particulate immobilized antibody. 2. The method according to claim 1, characterized in that the biological Liquid is removed from the test plate after the predetermined time and this is rinsed. 3. The method according to claim 1 or 2, characterized in that a Test plate with a protein A and / or protein G as an adherent bin is used. 4. The method according to any one of the preceding claims, characterized indicates that a test plate is used which is coated with an avidin, Streptavidin or derivatives thereof provided as an adhesive binding agent is what this binding agent with a biotin-labeling antigen or antibody is reacted.