JPH0752195B2 - Atssay method using bound members on particles and on filters or membranes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and kits for specific binding assays, such as immunoassays, using binding members on suspendable particles.

Immunoassay methods using suspendable particles (eg polystyrene latex) are well known, where the presence of analyte binding members affects the binding of the particles to each other.
In the case of manual assays, aggregated particles are observed or separated by specific gravity. U.S. Pat. No. 4,202,665 (Wentz et al., 1980) and pending U.S. patent application 733,68.
In case of No. 8 (Yensen et al.), The aggregated particles behave specially in the centrifugal field. Pending US Patent Application No. 733,689
In the case of No. (Lentrichia et al.), Two particles with different specific gravities bind to each other in an amount affected by the analyte concentration. When the first particles having one of the specific gravities move due to the centrifugal action, the bound second particles of the other one move together with the first particles. See also U.S. Pat. No. 4,115,535 (Diaber, 1978) for an assay method involving two particles each of which is protein bound to the same analyte.

U.S. Pat. No. 4,459,361 (Gefter, 1984) describes a particle immunoassay method in which particles bind or aggregate with other particles as a function of analyte ligand concentration. By passing (eg by a filter with a pore size of 0.4 μm) single particles (eg with a diameter of 0.3 μm) pass through, while duplex particles and higher particle agglomerates are captured by the filter. It
Particles passing through the filter can be visualized or enhanced by the optical density, fluorescence or other properties of the particles or by the enzymes attached to the particles (4
Column 42 line-5 column 4 line).

U.S. Pat. No. 4,424,279 (Bourne et al., 1984) discloses an assay method and a plunger in which large particles (20-30 μm in diameter) bearing binding members (eg, antibodies) are placed in the instrument compartment. The device is shown.
The analyte binding member (eg, antigen) and the soluble labeled binding member (eg, second antibody-enzyme) each enter the device compartment during the reaction period, and then the liquid is removed from the device compartment by vacuum to remove the particles. Are retained by a coarse (eg, pore size 15-17 μm) filter. The particles remaining in the device compartment are then assayed for label by, for example, adding an enzymatic reagent and cleaving the bond between the enzyme and the second antibody.

Ame Blankstein's candy. Clini. Proda. Levi. (Am.Clin.Prod.Rev.) 33-41 (November 1985)
And U.S. Pat. No. 4,200,690 (Root et al., 1980),
Several publications, including 4,246,339 (Cole et al., 1981) and 4,407,943 (1983), have shown that small molecules, or small molecules and bacteria, are large enough to translocate across the membrane. An immunoassay method for immobilizing an antibody or an antigen on a microporous membrane having a pore size (0.65 to 5.0 μm) is described. For example, in U.S. Pat.No. 4,200,690,
Antibodies coated with this type of membrane capture the antigen that is the analyte from the crude sample. After washing, an enzyme-bound second antibody (soluble conjugate) is introduced and specifically binds to the bound analyte antigen. After washing, the enzymatic readings are compared to the values obtained from a similar membrane containing an antibody that is not specific for the analyte. U.S. Pat.No. 4,407,943 (Cole et al., 198
3 years) is the same.

U.S. Pat. No. 4,486,530 (David et al.) Shows an immunoassay method using a sandwich type monoclonal antibody. However, in column 15, lines 33-51, a chromophore-labeled antigen and a latex-bound monoclonal antibody were used to inhibit the analyte antigen from aggregating, thus leaving the chromophore in solution, where the chromophores remain. It describes the Atsushi method that does not quench the light. Another inhibitory assay, in which one binding member is particle bound, is described in US Pat. No. 4,308,206 (Mochida, 1981).

In the present invention, the first reagent-based binding member reacts with the sample, thereby binding the analyte-based binding member to the first reagent-based binding member. The reaction mixture is then passed through a filter or membrane that has a pore size sufficient to allow the particles to pass through. The second reagent-based binding member is immobilized on a filter or membrane and preferentially captures either particles with or without bound analyte-based binding member. The other type of particles remains in the state of passing through the filter, and the resistive pulse technique (resistive pulse techniqu
e) is detected by enzymatic reading or by absorption or scattering of light.

Accordingly, the present invention provides (a) suspended particles having a first reagent system binding member complementary to the analyte system binding member, when the analyte system binding member is present, this is the first reagent. Contacting with a biological sample under conditions that bind the system-bound member, and (b) contacting the filter with a pore size large enough to pass through the filter if the particles do not bind to the filter. Conducting the reaction mixture of (a); wherein the filter carries a second reagent-based binding member complementary to either the analyte-based binding member or the first reagent-based binding member; and (C) detecting particles that have passed through the filter as an indicator of the presence and amount of analyte bound binding members in the biological sample, the detection of the analyte bound binding members in the biological sample. Provide the law.

The present invention comprises: (a) a suspension of particles carrying a first reagent-based binding member complementary to the analyte binding member, and (b) if the particles do not bind to the filter, pass through the filter. A filter having a pore size large enough to allow for: (c) a second immobilized on the filter that is complementary to either the analyte-based binding member or the first reagent-based binding member. It also provides a kit for measuring (qualitatively or quantitatively) the analyte binding member in a biological sample, the kit comprising a reagent binding member.

The present invention finds use in the qualitative or quantitative detection of various binding members, including antigens, haptens, antibodies, binding proteins, receptors, and nucleic acid sequences. Because of the ease of presentation, an assay for antigens or antibodies will be described first. A description of the assays for other analytes follows. The term "complementary" with respect to first and second binding members is used in the sense that one is generally capable of specifically binding one to the other by ionic interactions and / or hydrogen bonds. . The term "competitive" with respect to the first and second binding members is each complementary to the third binding member, and the first binding member to the third binding member. The third bond which the second bond member can bond by the bond of
The sites on the binding member are reduced (or lost),
Alternatively, it is used in the sense that the binding between the second binding member and the third binding member is suppressed (for example, sterically). The term "sandwich" allows the third binding member to bind simultaneously to the first binding member and the second binding member, which generally results in the absence of significant steric hindrance between them. Is used to mean that crosslinks are formed.

The two reagents used in the present invention are a suspension of particles carrying a first binding member and a filter or membrane carrying a second binding member. Particles are of various cellular types; of synthetic or inorganic type, eg fixed red blood cells, bacterial cells, virus particles, polystyrene latex particles,
These are metal sols, dyed cholesterol-lecithin particles, charcoal, granular pigments, and bentonite particles. Preferred are synthetic polymer latex particles, especially polystyrene latex particles. Particles of this type are of various dimensions, for example diameters of 0.05 to 5.0 microns, diameters of 0.1 to
1.5 micron particles are preferred. Particles with a relatively narrow size distribution are used (eg 90% of particles have a median diameter of 10
%, But a slightly wider distribution is acceptable.

The first binding member can be attached to these particles by various known methods,
For example, it can be adsorbed or bound by physical adsorption, carbodiimide chemical bonding, or crosslinking with monofunctional or difunctional aldehydes. These particles may be pre-coated or simultaneously coated with a substance such as albumin to prevent non-specific adsorption or aggregation of the particles. These particles are further labeled, such as fluorescent molecules,
It may contain enzymes, dyes or radioisotopes, or they may be adsorbed or bound. In the system exemplified below, the first binding member is an antibody specific for digoxin.

In addition to attaching the label (eg enzyme) directly to the particle, the label is attached to the binding member (ie anti-digoxin antibody) or the label (eg enzyme) is attached to one of avidin or biotin, Attaching the other of avidin or biotin to a binding member (eg anti-digokin antibody) or to a particle (by covalent or adsorption) and thereby indirectly attached via an avidin-biotin bridge. You can also

The reagent containing the second binding member is a membrane or filter having the second binding member bound thereto. Membranes or filters of this type are often used for two purposes: (1) affinity purification of complementary binding members, or (2) diagnostic assays for complementary binding members in which the remaining reagents are dissolved. It is reported to be used for. Particularly preferred are US Pat. Nos. 4,066,512, 4,200,690, 4,24.
The microporous membrane described in 6,339 and 4,407,943. In these cases, a polymeric membrane (eg, nylon or poly (vinylidin fluoride)) is coated with both a second binding member and an inactive protein (eg, zein) to delay non-specific adsorption. For the present invention, the pore size of the membrane should be larger than the diameter of the particles, preferably at least about 400% of the average diameter of the particles.
For example, with a membrane having a pore size of 5.0 microns, particles having an average diameter of less than 5.0 microns can be used, but particles having an average diameter of about 1.25 microns or less are preferred. At the same time, when using a membrane with a pore size of 3.0 microns (filter), the particles preferably have an average diameter of 0.75 microns or less.

As an example, in the case of digoxin (a hapten that is often monitored as a therapeutic agent) assay, the particles carry anti-digoxin antibodies (produced by inoculating animals, eg rabbits with digoxin conjugates, eg digoxin-albumin). You can. In this case, the membrane may carry the digoxin-albumin conjugate. After the particles come into contact with the sample (eg, obtained from the patient's serum), some particles will have antibody sites blocked with the analyte digoxin. The reaction reaches equilibrium very quickly, and in most cases less than 1 minute is required to reach equilibrium or react to a sufficient degree. The more abundant analyte digoxin will be, the more or less part of the antibody site of the particle will be blocked in this way. When the reaction mixture is then passed through a membrane, particles with little or no free antibody sites preferentially pass through the filter; particles with most or all of their antibody sites are free of membrane digoxin. -Attached to the albumin site,
Will not pass. This reaction is also very fast and can be carried out simply by passing the reaction mixture through a filter by the action of pressure or vacuum. Digoxin levels in serum (over the desired range) if antibody loading on the particles is adequate and particle / serum levels are appropriate (each easily determined by lutein experiments). And an assay system that provides a measurable positive correlation between particles in the eluate that has passed through the membrane. The examples below show dose-response curves for such cases. Eluted particles were measured by enzymatic readout (Example 1), turbidity measurement (Example 2), or by particle counting method (performed using the same latex reagent and treatment filter as described in Example 2). It was

Some other systems according to the present invention find use in the present invention to detect antibodies, nucleic acid sequences or multiepitopic antigens. For example, for the detection of antibodies, a complementary antigen can be applied to the particles and an antibody that competes with the antibody to be analyzed can be applied to the membrane (filter). For detection of the target nucleic acid sequence, a complementary nucleic acid sequence is
Competing nucleic acid sequences can be applied to the membrane (see pending US patent application Ser. No. 790,671, filed Diamond and Collins, filed Oct. 23, 1985). For the detection of multivalent antigens (including, for example, Epitope-1 and Epitope-2), anti-Epitope-1 antibody is applied to the particles and Anti-Epitope-2 antibody is applied to the membrane to form Sangeriti. As the amount of the analyte multi-epitope type antigen increases, the amount of carried particles also increases.

Similarly, embodiments of the invention of the Saint-Germany type or the competitive type can be adopted for detecting the following. Haptens (including digoxin, theophylline, thyroxine, drugs of abuse, and gentamicin), antigens (including HCG, LH, transferin, CRP and CEA), viral antigens (eg common or special types of herpes) , Common or special types of hepatitis, and HTLV-III), tumor antigens, exotoxins, other proteins excreted by bacteria or parasites, antibodies (streptolidine-O, DNase-B, HTLV-III).
Or rubella, or those associated with infectious mononucleosis), viral DNA or RNA sequences, bacterial or parasitic DNA, mRNA or rRNA sequences, folate binding proteins or thyroxine binding proteins.

Generally, it is preferred that the binding member on one particle does not specifically bind to the binding member on another particle.

Example 1 A circle having a diameter of 13 mm was cut from 929 cm ² (5 ft ² ) of an immunoaffinity membrane "Biodyne"(# BIA050C5, Paul Corporation) having a pore size of 5.0 μm.
The carboxylate-activated filter is then washed with PBS
10 mg / ml solution of BSA in PBS or 10 mg / ml solution of BSA-digoxin conjugate in PBS was immersed for 2 hours at room temperature. The filters were then similarly washed by incubating in PBS alone. After moisture absorption, the BSA and BSA-digoxin activated filters were air dried.

0.30 micron diameter unmodified polystyrene latex particles (Nippon Synthetic Rubber Co., Ltd., # G2203) were first mixed with anti-digoxin rabbit whole serum at a protein content of 180 μg / latex mg in 0.05 M phosphate buffer (pH 7.8) With gentle stirring at room temperature in
It was adsorbed by incubating for 2.5 hours. After washing the latex by centrifugation once in the same phosphate buffer, the partially adsorbed particles are then purified glucose oxidase (EC 1.1.3.
Incubated in a 64 mg / ml solution of 4.) for 10 hours at 4 ° C. After repeated washing of the latex by centrifugation in PBS containing 10 mg / BSAml (PBS / BSA), the particles are suspended in the same buffer to a final concentration of 20 μg / ml (0.002%) of the latex. did.

The antigen sensitized filter was inserted into a Swinnie type holder equipped with a 1 cc syringe. The above-mentioned Latex reagent (0.25 ml of 0.002%) was mixed with 0.25 ml of PBS / BSA which seems to contain various concentrations of the analyte, and the whole mixture was passed through a filter, glucose 1.8%, horseradish was added. It was introduced into a test tube containing 0.30 ml of a substrate mixture containing 8.7 units / ml of peroxidase and 1.2 mM of 4-aminoantipyrine. The entire mixture was stirred and incubated at 37 ° C for 5 minutes and the absorbance read at 510 nm.

Digoxin-sensitized filters account for approximately total enzyme activity detected.
While retaining 80%, the BSA sensitized filter retained only about 28% (Table I). This difference reflects the number of latex particles specifically bound to the filter by digoxin immunoreactivity. The number of particles retained can also be detected by incubating the filter itself with the substrate mixture.

Sensitivity Latex particles (0.49 μm) were adsorbed with anti-digoxin serum and glucose oxidase as described above. Then 0.25 ml of 0.0132% (w / w) latex suspension was added to 0.25 ml of PBS / BSA buffer containing various concentrations of digoxin.
And then to the digoxin sensitizing filter,
The substrate solution was introduced into 0.30 ml (total volume 0.80 ml). The reaction mixture was mixed and incubated at 37 ° C for 5 minutes, then 51
The optical density at 0 nm was recorded.

The assay was sensitive to at least 5 ng / ml (Table II). Only 7% of total activity due to non-specific interactions
Were retained on the membrane (unovercontrol vs. BSA over).

Example 2 1.09 micron diameter unmodified polystyrene latex particles (Nippon Synthetic Rubber, # G2101) with anti-digoxin rabbit whole serum at 200 μg protein / mg latex are under the same conditions as described in Example 1. Adsorbed. After particle sensitization with this antibody, they were washed by repeated centrifugation in PBS / BSA and finally resuspended to a concentration of 0.003% (w / w) as measured by optical density at 650 nm. "Biodyne", an immunoaffinity membrane with an average pore size of 5.0 microns (# BI050C5, Paul Corporation)
Was cut into a circle with a diameter of 13 mm and activated with the BSA-digoxin conjugate as described in Example 1.

1.5 ml of a 0.003% suspension of Latex to perform an assay
Was mixed with 0.5 ml of a solution containing digoxin in PBS and the mixture was
A 5cc syringe was used to conduct the immunoaffinity membrane. The optical density of the liquid was then measured spectrophotometrically at 650 nm.

As shown in Table III, the OD in the presence of 10 ng / ml of analyte was 7 times greater than that observed in the absence of digoxin.
Was recognized. Moreover, the number of particles passing through the filter continued to increase with increasing digoxin concentration. 100ng / ml
, A 14-fold increase was observed. This is a concentration close to saturation for the particle-immobilized binding site.

Example 3 Preparation of Reagents An immunoaffinity filter "Biodyne" (pore size 5.0 μm) was first sensitized with human chorionic gonadotropin (HCG) by the following method.

Approximately 35 filters with a diameter of 13 mm are separately
HCG purified by DEAE ion exchange chromatography
(8409 IU / mg solid) 2 ml solution containing 4.5 mg / ml in PBS
I put it in. After soaking at room temperature for 1 hour, bovine serum albumin (BSA) 10 mg / ml and sodium azide 0.1
Transfer the filter to 250 ml of PBS solution containing
Soaked for 20 minutes. The filter was rinsed by a final immersion in distilled water, absorbed on paper and dried at room temperature.

Latex particles (JSR # U0403) with a diameter of 0.49 μm were sensitized with anti-β-HCG monoclonal antibody and glucose oxidase by the following method. That is, particles washed in 0.01M sodium phosphate buffer (pH 7.8) were suspended in the same buffer to obtain a final concentration of about 10%, and 20 mg (0.20 m
l) was transferred to a small glass screw cap bottle. Then, 0.10 ml of purified monoclonal anti-β-HCG antibody (Maritime Chemical Co., Ltd.) (0.56 mg of protein) was added. After incubating overnight at 4 ° C, the latex sample was washed once by centrifugation in phosphate buffer, resuspended in 0.10 ml of the same buffer, and purified solid glucose oxidase (Boehringer Mannheim) 10 mg.
Was added to make the total volume about 0.20 ml. The reaction mixture was incubated at room temperature for 1 hour, washed twice with 10 mg / ml BSA in PBS by centrifugation and PBS / BSA containing 0.1% azide.
Resuspended in 0.88% final (latex 8.8mg / ml)
I said.

Atsushi procedure Prepare a fresh substrate mixture as described in Example 1, 0.5 ml.
Was placed in each of 5 12 × 75 mm glass test tubes. The above HCG sensitized filters were each inserted into a Sweeney type filter holder equipped with a 1 cc tuberculin syringe (without a plunger). The latex-analyte mixture contained 0.01 ml of 0.88% latex and PB as shown in Table IV.
Solutions containing various concentrations of purified HCG in S / BSA buffer.
It was prepared by mixing with 69 ml. 2-3 at room temperature
After standing for a minute, the latex suspension was separately transferred to different syringes with filters and fed into test tubes containing the substrate.

The absorbance of each suspension at a wavelength of 510 nm was measured by incubating for 5 minutes in a 37 ° C. water bath and then inserting the test tube directly into a Spectronik 21 type spectrophotometer (Bauille and Rom).

The standard curve for HCG by the above method is shown in Table IV.
The maximum part of the total difference in the obtained OD was observed at a concentration of 5 IU / ml (84%), and the OD up to 40 IU / ml gradually increased.

Claims (15)

[Claims] 1. (a) Suspended particles having a first reagent system binding member complementary to the analyte system binding member, when the analyte system binding member is present, this is the first reagent. Contacting with a biological sample under conditions that bind the system-bound member, and (b) contacting the filter with a pore size large enough to pass through the filter if the particles do not bind to the filter. Conducting the reaction mixture of (a); wherein the filter carries a second reagent-based binding member complementary to either the analyte-based binding member or the first reagent-based binding member; and (C) detecting particles that have passed through the filter as an indicator of the presence and amount of analyte bound binding members in the biological sample, the detection of the analyte bound binding members in the biological sample. Law. 2. A second reagent-based binding member that is complementary to the first reagent-based binding member and competes with the analyte binding member for a binding site on the first reagent-based binding member. The method according to claim 1. 3. The method according to claim 2, wherein the first reagent system binding member is an antibody. 4. The method according to claim 3, wherein the second reagent system binding member and the analyte system binding member are haptens. 5. The method according to claim 3, wherein the second reagent system binding member and the analyte system binding member are antigens. 6. The binding member of the analyte binding member different from the binding site of the analyte binding member binding to the first reagent binding member. The method of claim 1 which is complementary to the analytical system binding partner. 7. The analyte binding partner is an antigen having at least two distinct epitopes and the first and second reagent binding members are antibodies specific to different epitopes of the antigen. A method according to claim 6. 8. The method according to claim 6, wherein the analyte binding member is an antibody. 9. The first reagent system binding member is an antigen or a hapten, to which the analyte antibody is specific,
9. The method of claim 8, wherein the second reagent system binding member is an antibody specific for the analyte system antibody or for the immune complex containing the analyte system antibody. 10. The method of claim 1 wherein the particles are 0.05 to 5 microns in diameter and the filters are of a pore size of at least 200% of the particle diameter. 11. The method according to claim 1, wherein the detecting step (c) comprises conducting the eluate passed through the filter to the orifice and measuring the impedance passing through the orifice.
The method described in the section. 12. The method according to claim 1, wherein the detecting step (c) comprises measuring the absorbance of the eluate that has passed through the filter. 13. The method of claim 1 wherein the particles are fluorescent and the detecting step (c) comprises measuring the fluorescence of the eluate that has passed through the filter. 14. The method according to claim 1, wherein the particles carry an enzyme, and the detecting step (c) comprises mixing the eluate passed through the orifice with a reagent for the enzyme. 15. (a) A suspension of particles carrying a first reagent-based binding member complementary to the analyte binding member, and (b) if the particles do not bind to the filter, pass through the filter. A filter having a pore size large enough to allow for, and (c) a first group immobilized on the filter that is complementary to either the analyte-based binding member or the first reagent-based binding member. A kit for measuring an analyte binding member in a biological sample, which comprises two reagent binding members.