GB2324601A

GB2324601A - Solid phase plate assay using an immunoreagent immobilized on particles as a detector system

Info

Publication number: GB2324601A
Application number: GB9804529A
Authority: GB
Inventors: Mikio Nakayama; Ayumi Mito
Original assignee: Asahi Kogaku Kogyo Co Ltd
Current assignee: Pentax Corp
Priority date: 1997-03-03
Filing date: 1998-03-03
Publication date: 1998-10-28
Also published as: GB9804529D0; DE19808930A1; JPH10246725A; CA2230773A1

Abstract

A multi-well test plate is coated with a binding agent which binds a candidate analyte in a biological sample. The plate is then reacted with particles bearing an immunoreagent which binds to the analyte immobilized on the plate. Aggregation of particles indicating a positive reaction is assessed visually. In one embodiment protein A or protein G is immobilized on a plate and a serum sample is subsequently added. After rinsing, particles containing paragonimiasis antigen are added to the plate to detect corresponding serum-derived antibodies bound thereto. In another embodiment avidin is immobilized on plates and biotin conjugated anti-human IgG bound thereto. The assay then proceeds in the same way as for the protein A assay. Particles comprising a ceramic/polymer composite such as hydroxyapatite on nylon beads are described.

Description

2324601 METHOD FOR THE DETECTION OF ANTIGENS OR ANTIBODIES The present

invention relates to a method for the detection of an antigen or antibody in a biological fluid such as saliva, blood, lymph, faeces, urine by using an aggregation reaction method based on an antigen- antibody reaction.

Hitherto, an aggregation reaction method using aggregating composite particlesp such as gelatin, kaolin, synthetic polymer with the immobilized antigenlhas been used in the determination of an amount of the specific antibody in a test medium such as blood and body f luids. In this aggregation reaction method, detection and judgement are made by employing a test plate with a plurality of wells which are used to prepare serial dilution of the test medium. In the detection process, if the aggregation reaction arises (for a positive reaction), a mat-like aggregation image is formed as uniformly distributed aggregate on the walls of the wells, whereas if no aggregation reaction arises (for a negative reaction), a circular button-like precipitate - 2 is deposited at the bottom of the wells, since the precipitate runs down from the walls of the wells.

However, in the tests concerning the hemagglutination inhibition of the prior art, it was necessary to further include the troublesome operation of removing an inhibitor, i.e. substances capable of inhibiting an antigen-antibody reaction, such as lipid, glycoprotein in the serum. Furthermore, such an inhibitor -removal process generally required 24 hours of operation.

An object of the present invention is to provide a method of detecting an antigen or antibody in a biological fluid which enables easy detection of the antigen or antibody with a high level of accuracy without requiring an inhibitor removal operation.

According to the present invention there is provided a method for the detection of an antigen or antibody in a biological fluid to be tested, which comprises:

applying the biological fluid to a test plate for an aggregation test having, adhered to a surface thereof, a binding agent for the antigen or antibody to add the biological fluid to each of wells of the test plate; preferably, the discarding of the biological fluid from the test plate and rinsing the test plate, after retention of the biological fluid in the wells for a predetermined period of time; and adding a liquid containing particles of the immobilized antigen or antibody to the wells.

That is, the present invention is based on the findings that the above object can be accomplished by adding a test medium to each of the wells of the test plate for an aggregation test having adhered to a surface thereof a binding agent for an antigen. or antibody to thereby bond an antigen or antibody in the test medium and the binding agent, discarding the remaining test medium from each well of the test plate, and washing the test plate to remove a remainder of the adhered inhibitor and others from a wall surface of the test plate.

Using the detection method of the antigen or antibody according to the present invention, it becomes possible to omit the operation for removing an inhibitor from the detection process using the aggregation reaction method, prevent a formation of a pseudo-aggregation image which is observed in, for example, the dilution of the test medium such as serum at a low dilution degree, and also it enables an easy detection and judgement with a high level of accuracy.

The present invention will now be described below.

A test plate for an aggregation test used in the present invention is not restricted, as long as it has a plurality of wells for use in the preparation of serial dilution of a test medium. Generally, the test plate may be a plate or strip with wells, and a bottom portion of each of the wells has a V-shaped, U-shaped or similar cross-section or a modification thereof. The test plate may be preferably produced from a plastics material, and typical examples thereof, although not restricted to, include polystyrene, polypropylene, polyvinyl chloride.

in the present invention, the above-described test plate is used after a binding agent for an antigen or antibody was adhered to a bottom portion of each well of the test plate, that is, an inner surface of the V-shaped, U-shaped or similar cross-sectional portion of each well of the test plate. For example, the test plate may be used after an inner surface of each well of the test plate is adhered with protein A and/or protein G, or after the inner surface of each well of the test plate is adhered with an avidin, streptavidin or derivatives thereof, followed by reacting the avidin, streptavidin or derivatives thereof with a biotin-labeling antigen or antibody. Suitable examples of the avidin derivatives include NeutraAvidin (commercially available from Pierce Inc.) and UltraAvidin (commercially - 5 available from Leinco Technologies Inc.).

In the practice of the present invention, a biological fluid to be tested, i.e., test medium, is first contained in each well of the test plate f or an aggregation test. The test plate used has a plurality of wells for receiving the test medium, and contains a binding agent for the antigen or antibody adhered to a surface thereof. in each well, an antigen or antibody contained in the test medium is bonded to the binding agent. To obtain a satisfactory bonding effect, it is preferred that the test plate with the added test medium is left to stand or shaken for a predetermined amount of time. The standing or shaking time is generally in the range of about 15 minutes to about one hour, but preferably at least 20 minutes. Shaking preferably is applied to attain an effective bonding between the antigen or antibody and the bonding agent.

According to prior art methods, after the addition of the test medium to the test plate and while retaining the test medium in each well, particles of the immobilized antigen or antibody to be detected (prepared by immobilizing on a particulate carrier) are added to the wells of the test plate to cause an antigen -antibody reaction. However, the antigenantibody reaction is inhibited in these methods, because an inhibitor contained in the test medium, such as lipid or glycoprotein, can cover the particulate immobilized antigen or antibody.

Conversely, with the present invention, such inhibition of the antigen-antibody reaction can be prevented, because, in accordance with the method of the present invention, a test medium is added to each well of the test plate and the added test medium is preferably discarded from the test plate after the retention of the test medium over a predetermined period of time, followed by the rinsing of the test plate to remove all the substances other than the antibody or antigen bonded to the bonding agent therefor. The rinsing of the test plate may be carried out by rinsing the wells, preferably the vacant wells after discarding of the test medium, with water, a physiological salt solution, or a physiological saline. With rinsing, the remaining inhibitor of the test plate can be removed, thereby ensuring omission of the subsequent inhibitor removal which is essential as a specific process to the prior art methods.

After rinsing of the test plate, the particular immobilized antigen or antibody is added to the wells of the test plate. If a trace amount of the test medium remaining on a wall surface of the wells and contains an antibody or antigen to be detected, an antigen-antibody reaction can be generated with a good accuracy, and thus an antibody or - 7 antigen in the test medium can be detected with high accuracy. Mat- like aggregation images are produced if any antibody or antigen is contained in the test medium.

The particulate immobilized ant'lgen or antibody that can be used in the detection of an antigen or antibody by the present invention is not restricted to a specific one. Preferably, the immobilized antigen or antibody is used as aggregating composite particles which comprise an antigen or antibody immobilized on a composite body of ceramics and polymer, latex, gelatin or kaolin. Aggregating composite particles composed of a composite body of ceramics and polymer having immobilized thereon an antigen or antibody include, for example, those disclosed in Japanese Unexamined Patent Publication (Kokai) No. 7-174762 and U.S. Patent No. 5,540,995 which are incorporated by reference herein in their entireties. That is, those prepared by adsorbing and immobilizing an antigen or antibody onto a particulate composite body consisting of polymer particles having coated on a surface thereof a calcium phosphate compound, the polymer particles being dyed or the whole composite body being dyed, followed by treating unadsorbed sites of the composite body with a blocking agent. Preferably, used as the particulate composite bodies used are those prepared by physically striking particles of the calcium phosphate - 8 compound against the polymer particles, thereby coating a surface of the polymer particles with the calcium phosphate compound.

With the above-described calcium phosphate compound, a wide variety of calcium phosphate compounds may be used, as long as they show a Ca/P ratio in the range of 1. 0 to 2.0. For example, one member or a combination of two or more members selected from the group consisting of Calo(P04)6(0H)2, Cajo(P04)6F2, Calo(P04)6C12e Ca3(PM2e Ca2P207. Ca40(PM2 and CaHP04 may be used as the calcium phosphate compound. Among these calcium phosphate compounds, hydroxyapatite and tricalcium, phosphate are preferably used, and the most preferred one is a calcium phosphate compound which contains hydroxyapatite as a principal component thereof. When fluoroapatite is used as the calcium phosphate compound, it is preferred that the content of fluorine in all the calcium phosphate compounds used is not more than about 5 % by weight, because a fluorine content above about 5 % by weight can cause an eluation of fluorine from such compounds. These calcium phosphate compounds may be produced in accordance with any one of the well-known production methods including a wet synthesis process, a dry synthesis process and others.

The particles of the calcium phosphate compound can be produced, for example, by spray-drying a slurry of the - 9 calcium phosphate compound to obtain particles thereof, followed by firing the obtained calcium phosphate particles. Alternatively, in addition to this production process, any other granulation methods may be used to produce similar calcium phosphate particles. Preferably, sieving and other separation methods may be used to selectively obtain the particles of the calcium phosphate compound having the predetermined range of particle size depending on the intended use of the particles.

EXAMPLES

The present invention will be further described with reference to the examples thereof. Note, however, that the present invention should not be restricted to these,=xamples. Moreover, all parts and percentages in the examples are by weight unless indicated otherwise.

Preparatory Example 1:

Production of Composite Particles of Ceramics and Polymer with Immobilized Japanese Encephalitis Virus of Beijing Strain.

50g of nylon beads having an average particle diameter of about 5 ti m dyed with an anthraquinone dye, commercially available under the trade name "Mitsui ML Colors ML red VF21, from Mitsui Toatsu Senryo Co., and 7.5g of hydroxyapatite - 10 particles were blended for about 5 minutes at 320C to 500C in the Nara Hybridization System NHS-1 rotating at 8,000 rpm to coat a surface of the nylon beads with hydroxyapatite. In the thus obtained composite beads, the layer of hydroxyapatite coating had an average thickness of 0.44/im and an average particle diameter of 5.8 tLm.

32ttg/mL of Japanese encephalitis virus of Beijing strain and a 10% (w/v) solution of the composite beads were mixed in an equivalent ratio, and kneaded for about one hour in a rotator to cause an adsorption of the virus antigen on the composite beads. The composite beads were centrifuged for about 5 minutes at 1,000 rpm in a centrifugal machine to precipitate pellets, while the simultaneously produced supernatant was discarded. The pellets were loosened, and the antigen was immobilized on the pellets with 0.05% of glutaraldehyde as a diluted solution of phosphoric acid buffer solution having a pH value of 7.2 (hereinafter, referred to as "PBS"). After stirring at room temperature in a rotator, the beads with the immobilized antigen were centrifuged for about 5 minutes at 1,000 rpm in a centrifugal machine to precipitate pellets, while the simultaneously produced supernatant was discarded. Using 1% (w/v) solution of the masking agent commercially available under the trade name "Block Ace" from Daini-pPon Seiyaku Co., - 11 masking was made for about one hour to mask antigenunadsorbed sites of the beads, while the beads were stirred in a rotator. After stirring in a rotator at room temperature, the beads were centrifuged for about 5 minutes at 1,000 rpm in a centrifugal machine to precipitate pellets, while the simultaneously produced supernatant was discarded. To prevent the release of the Block Ace from the beads, immobilization was made using 0.05% of glutaraldehyde, diluted solution of PBS (pH= 7.2). After stirring at room temperature in a rotator, the beads were centrifuged for about 5 minutes at 1,000 rpm in a centrifugal machine to precipitate pellets, while the simultaneously produced supernatant was discarded. Using 1% (w/v) solution of Block Ace, masking was made to inactivate active residues of the glutaraldehyde, followed by keeping the beads in a 0.4% (w/v) solution of Block Ace.

Preparatory Example 2: Production of Aggregation Test Plate 0.05mL of the PBS solution containing a protein A at a concentration of 1. 0 X 10-3 Mg/ML was added to each of the wells of the polystyrene plate. The polystyrene plate used herein contained 96 V-shaped wells having a capacity of 0.3mL. After one minute, to remove the liberating protein A in the PBS solution of the wells, the PBS solution was - 12 discarded from the polystyrene plate, 0.05mL of the PBS solution was added to each of the vacant wells of the plate, and the PBS solution was again discarded. The plate was dried to obtain a test plate having the adhered protein A.

Preparatory Example 3:

Production of Composite Particles of Ceramics and Polymer with Immobilized Paragonimiasis Antigen The procedure of the Reference Example 1 was repeated to produce composite particles of ceramics and polymer with immobilized paragonimiasis antigen with the proviso that 25 4g/mL, based on an amount of the antigen protein, of the paragonimiasis antigen was used in place of the Japanese encephalitis virus of Beijing strain.

Example 1:

gL of serum (test medium), from a Japanese encephal itis patient, diluted with PBS to make ten-times more volume, was added to each of the wells of the first column of the protein A-adhered test plate produced in the Reference Example 2, and 50 JIL of the PBS solution containing 10% of Block Ace was added to each of the wells of the remaining columns of the test plate. Then, dilution was made from the first column of the test plate using 50 IL L of PBS in each dilution in a dilution factor of 2 n, 13 - i.e., two-fold serial dilution. After the test plate was shaken for about one hour, the test medium was removed from all the wells, and the test plate was washed with water, followed by draining water from the plate. Thereafter, 100 J.LL of the 0.1% solution of the composite particles of the ceramics and polymer with the immobilized Japanese encephalitis virus of Beijing strain produced in the Reference Example 1 was added to the vacant wells of the drained test plate. After about two hours, it was observed that clear positive images could be produced from the serum dilution factor of 10 times.

Example 2:

(1) Production of the Plate for the Determination of IgG Specific to the Paragonimiasis 504L of the solution obtained upon dilution of the avidin, commercially available under the trade name "NeutraAvidin" from Pierce Co., with a carbonate buffer solution (PH = 9.6) to make 25 /4g/mL was added to each of the wells of the microtitration plate commercially available from Glynor Co. The microtitration plate used herein contained 96 (12X8) V-shaped wells. The solution was discarded from the plate, after the plate was left to stand for about one hour at room temperature. In the obtained avidin-immobilized plate, each well was washed with deionized water, and then filled with 10OgL of four times- diluted solution of Block Ace (in PBS, 1% by weight per solution). The plate was left to stand for one hour at room temperature, and then the diluted solution of Block Ace was discarded from the plate. The plate was air-dried to obtain an avidin-immobilized plate.

After O.Smg/mL of antihuman IgG biotin-labeling antibody was diluted to make 400-times the volume thereof, each 509L of the diluted solution was added to each well of the avidinimmobilized plate. The plate was left to stand for about one hour at room temperature to induce the bonding reaction between the avidin and the biotin. A plate for the determination of IgG specific to the paragonimiasis was obtained.

(2) Detemination of the Human Serum A human serum which is positive to the paragonimiasis and a human serum which is negative to the paragonimiasis each was diluted to make ten- times the volume. In the plate produced in the above procedure (1), the diluted serum was added to the first to 12th wells, and dilution was made in a dilution factor of 2n, i.e., two-fold serial dilution. In each of the wells, 50 9L of the diluted serum to be tested was contained. Under the same conditions, the plate was left to stand for about one hour at room temperature.

Thereafter, the plate was washed with deionized water, and then each well of the plate was filled with 100jUL of the 0.1% solution of the composite particles of the ceramics and polymer with the immobilized paragonimiasis antigen produced in the Reference Example 3. The plate was left to stand, and after one hour, the judgement was made to ascertain whether an aggregation image was formed or not. It was observed from the results that clear positive images could be obtained in the dilution range of from 10 times to 20480 times for the paragonimiasis-positive serum, and also clear negative images could be obtained in the dilution range of from 10 times to 20480 times for the paragonimiasisnegative serum.

Comparative Example 1 túL of the serum (test medium, same as that used in the Example 1) was added to each of the wells of the first column of the protein A-adhered test plate produced in the Reference Example 2, and 50 jUL of the PBS solution containing about 10% of Block Ace was added to each of the wells of the remaining columns of the test plate. Then, dilution was made from the first column of the test plate using 50 AL of PBS in each dilution in a dilution factor of 2 n, i.e., two-fold serial dilution, and the test plate was - 16 shaken for about one hour. Thereafter, 50 t4L of the 0.1% solution of the composite particles of the ceramics and polymer with the immobilized Japanese encephalitis virus of Beijing strain produced in the Reference Example 1 was added to each well of the test plate. After about two hours, it was observed from the judgement. that pseudo-negative reaction images could be produced in the serum dilution range of from 10 times to 80 times, and then positive reaction images could be produced with increase of the dilution factor over 80 times.

Although the invention has been described with reference to particular means, materials and embodiments, it is to be understood that the invention is not limited to the particulars disclosed and extends to all equivalents within the scope of the claims.

- 17

Claims

CLAIMS:-

1. A method for the detection of an antigen or antibody in a biological fluid to be tested, which comprises:adding said biological fluid to at least one well of a test plate, at least one well having adhered to a surface thereof a binding agent for the antigen or antibody; retaining said fluid in said at least one well for a predetermined period of time; and adding a liquid containing a particulate immobilized antigen or antibody to said at least one well.

2. A method for the detection of an antigen or antibody according to claim 1, in which said biological fluid is discarded from said test plate which is rinsed after said fluid has been retained in said at least one well for a predetermined period of time.

3. A method for the detection of an antigen or antibody according to claim 1 or 2 in which said test plate having a binding agent adhered to a surface thereof is a plate having adhered thereto protein A and/or protein G.

4. A method for the detection of an antigen or antibody according to any preceding claim in which said test plate having a binding agent adhered to a surface thereof is a plate - 18 prepared by adhering a plate with an avidin, streptavidin or derivatives thereof, followed by reacting said avidin, streptavidin or derivatives thereof with a biotin-labeling antigen or antibody.

5. A test plate f or the detection of an antigen or an antibody in a biological fluid to be tested:- said test plate comprising at least one well; characterized in that a bonding agent is disposed over a portion of the or each well in the test plate, said bonding agent being formed on the surface of the well by a test medium; and subsequently adding a liquid containing a particulate immobilized antigen or antibody to a said well.

6. A test plate according to claim 5 wherein the test medium is adapted to be discarded before addition of a liquid carrying the antibody or antigen.

7. A test plate according to claim 5 or 6 wherein the bonding agent adherent to the surface is selected from protein A and/or protein G.

8. A test plate according to any one of claims 5 to 7 prepared by disposing in at least one well an avidin, streptavidin or a biologically effective derivative thereof, whereby a biotin-labelled antigen or antibody can react therewith.

9. A diagnostic kit f or the detection of an antigen or - 19 antibody in a biological fluid to be tested; said kit comprising at least one well; characterized by a separate supply of a bonding agent for this disposition in the or each well.

10. A kit according to claim 9 wherein the antibody or antigen is immobilized, particulated and adapted for addition to the or each well.

11. A kit according to claim 9 or 10 wherein the bonding agent is selected from protein A and/or protein G.

12. A kit according to claim 10 wherein the bonding agent is avidin, streptavidin or a biologically effective derivative thereof, and wherein the immobilized antigen or antibody is biotin-labelled.

13. A method for the detection of an antigen or an antibody in a biological fluid to be tested substantially as hereinbefore set forth with reference to and/or as illustrated in, any one of the foregoing examples.

14. A test plate f or the detection of an antigen or an antibody in a biological f luid to be tested substantially as hereinbefore set forth with reference to, and/or as illustrated in, any one of the foregoing examples.

15. A diagnostic kit for the detection of an antigen or antibody in a biological f luid to be tested substantially as hereinbefore set forth with reference to, and/or as illustrated in, any one of the foregoing examples.