JPH0220067B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides shell-core particles in which the high refractive index of the core provides high sensitivity for light scattering measurements and in which the shell has functional groups capable of covalently binding compounds of biological interest. A new particle reagent consisting of: Compounds of particular biological interest are antibodies or antigens and the particle reagent is intended for use in light scattering immunoassays. Agglutination reactions have long been used for visual (semi-quantitative) and quantitative assays for a wide variety of bacteria, cell surface antigens, serum proteins, or other analytes of clinical interest. Agglutination results from the reaction between a bivalent antibody and a multivalent antigen of interest to produce an aggregate, which can be detected and/or measured in a variety of ways. Similarly, the same reaction can be used to detect specific antibodies by an agglutination reaction produced by addition of the corresponding antigen. In order to generate large cross-linked aggregates,
The number of reactive sites on the antigen must be two or more. Therefore, when detection of monovalent haptens was desired, the reaction scheme was modified as follows. That is, multivalent forms of the antigen, e.g. hapten-
A protein conjugate is generated and the hapten present in the sample competes with the multivalent form for effective binding sites on the antibody, thereby reducing the amount of aggregation. This technique is called aggregation inhibition. The production of polyvalent forms of haptens is old in the art. Often this hapten is conjugated to a carrier protein, as is done in the manufacture of immunogens. The stoichiometry of the reaction can be adjusted to include three or more haptens per protein molecule. The exact number will be determined by the requirements of the particular assay in which the material will be used. Increased sensitivity of visual or instrumental detection of aggregation or its inhibition can be achieved through the use of particle reagents as carriers rather than soluble proteins or protein conjugates. For example, antiserum against chicken egg albumin was shown to be 2000 times more sensitive in precipitating chicken egg albumin coated onto collodion particles than in precipitating chicken albumin itself [Immunology, Vol.
394 pages (1974)]. Antibody particle reagents are also known. A common method for producing such reagents is by adsorbing the antibody to the surface of a suitable adsorbent. Polystyrene-based latex particles are widely used for this purpose. However, these reagents undergo desorption during storage or use leading to variations in the properties of the reagents. This in turn can adversely affect the sensitivity and reproducibility of the assay. To overcome this desorption problem, particle reagents can be prepared by covalently attaching compounds of biological interest to the particle surface.
Polystyrene polymers are modified to include functional groups capable of covalent protein attachment.
US Pat. No. 4,064,080 discloses styrene polymers having terminal aminophenyl groups and proteins attached thereto. US Pat. No. 4,181,636 discloses xylated latex polymers coupled to immunologically active substances via water-soluble activators and their use as diagnostic agents in agglutination tests. US Patent No. 4210723
The specification has free epoxy groups on the particle surface.
Shell-core latex polymer particles with a diameter of 0.15-1.5 μm and the coupling of proteins through these epoxy groups are described. Other polymer systems have been developed for subsequent attachment of immunologically active substances. US Patent No.
No. 4,264,766 discloses latex polymers having a particle size of 0.01 to 0.9 μm and having carboxyl and amino groups to which active groups such as water-soluble polyhydroxy compounds can be covalently attached. Through the use of activating agents such as carbodiimides, immunologically active substances are bound to the latex particles/polyhydroxy compound carrier to produce diagnostically useful reagents. There is a need for stable particulate reagents of high purity for use in light scattering aggregation assays, which can conveniently be prepared by covalently attaching a compound of biological interest to a particulate carrier. There is. The particle reagents of the present invention have a high refractive index and essentially have (A) an inner core and an outer shell, where the inner core has a refractive index not less than 1.54 as measured at the wavelength of the sodium D line; and the outer shell has (1) an ethylenically unsaturated monomer having a functional group capable of reacting with a compound of biological interest selected from the group consisting of epoxy, carboxy, amino, hydroxy and aldehyde; ) optionally other ethylenically unsaturated monomers,
and (3) a polymer of not more than 10 polymerized parts of the inner core monomer of the outer shell, and the outer shell is produced by polymerization in the presence of said inner core from 0.03 to 0.1. (B) a compound of biological interest, an antigen thereof, or an antibody thereof, to which said polymer particle is covalently bound. Compounds of biological interest can be attached to the polymer particles directly or via protein substances. The method for determining compounds of biological interest of the present invention comprises (A)(1) essentially (a) having an inner core and an outer shell, wherein the inner core is at the wavelength of the sodium D line; and the outer shell (i) has a functional group capable of reacting with a compound of biological interest selected from the group consisting of epoxy, carboxy, amino, hydroxy and aldehyde; an ethylenically unsaturated monomer, (ii) optionally other ethylenically unsaturated monomers, and (iii) not more than 10 parts by weight of the inner core monomer of the outer shell; (b) the outer shell is made by polymerization in the presence of said inner core and has polymer particles having an approximate diameter range of 0.03 to 0.1 μm; and (b) said polymer particles are covalently bonded. (2) a liquid presumed to contain a compound of biological interest, and (3 ) culturing the flocculant; and (B) spectroscopically measuring the increased particle size resulting from the flocculation. The method of the present invention for protein determination can be carried out directly without aggregating agents using suitable complement particle reagents. The present invention relates to novel particle reagents with properties optimized for use in sensitive light scattering immunoassays. The particle reagent (1) is formed from a high refractive index core material, (2) has a shell material that can covalently bind a compound of biological interest, and (3) It is designed to maximize immunoassay sensitivity by having small particle size for optimal sensitivity in immunoassay. The light scattering properties of a particle suspension depend on several variables, most importantly the particle size, the refractive index of the core and suspending medium, and the wavelength of the light used for measurements. Thus, the selection of core material, particle size, and wavelength for agglutination detection are all important factors in optimizing assay sensitivity. These factors can be determined by the type of light scattering detection means used. During visual observation of the agglutination reaction, a broad wavelength band between approximately 400 and 650 nm is used. Since light scattering responsivity varies over this wavelength range, visual observation may result in averaging the effects of many less sensitive wavelengths rather than selecting the optimal wavelength for a given particle size and refractive index. Become. For particles small compared to the wavelength of light, scattering increases as the reciprocal of the fourth power of the wavelength and its magnitude depends on the refractive index. When the wavelength of the light approaches the absorption band of the particle, the refractive index increases and the light scattering properties therefore also become sensitive to the optical dispersion of the scattering element and the wavelength function can exceed the fourth power. For turbidimetric detection of particle size changes at a given measurement wavelength, it is important to choose particle size and refractive index carefully. This is because the turbidimeter signal passes through a maximum value with a dual value response with little or no sensitivity at the peak. Furthermore, the slope sensitivity is greater on the small particle size side of the peak than on the large side, and this increases as the particle to media ratio increases. For these reasons, high refractive index small particles with short wavelength detection are preferred for high sensitivity. There are practical limits to ultraviolet light for measuring samples in serum because of light absorption by proteins and other components. That is, the convenient wavelength is approximately
The wavelength is 320nm or more. Longer wavelengths can be used with lower sensitivity. Small particles, ie, those with a diameter of about 0.1 micron or less, are preferred for both increased slope sensitivity and reaction rate. For reasons of stability and convenience of synthesis, approximately 0.03 μm
A particle size of is preferred. Generally, a particle size range of 0.03 to 0.1 μm can be used in the particle reagents of the present invention. A shorter wavelength, eg 340 nm, will give a larger signal difference than a longer wavelength, eg 400 nm. For turbidimetric detection, the optimal sensitivity may depend not only on particle size and wavelength, but also on the measurement angle. Nephelometer refers to the measurement of light scattered at an angle from a beam of interest. The particle size for optimal sensitivity has angle dependence as well as wavelength dependence. Other types of light scattering measurements of aggregation reactions include particle calculations, quasi-elastic light scattering, self-correcting spectroscopy, and measurements of particle asymmetry or polarization. These types of measurements impose different limitations on particle reagents. However, in all types of measurements, the higher the refractive index of the particle at the chosen wavelength,
Light scattering signal is higher. A preferred method of measuring immune responses using the particle reagents of the present invention is by turbidity. The reason is that it does not require any special equipment other than a spectrophotometer commonly available in clinical laboratories. This spectrophotometer measures the increase in absorption due to the increase in particle size resulting from the aggregation reaction. This increased absorption is a direct measure of the aggregation caused by the analyte, or it is an indirect measure of the inhibition of aggregation caused by the analyte. Careful particle size selection is important in order to optimize the turbidity changes that occur during aggregation. During the aggregation reaction, the effective particle size increases. Therefore, in order to measure sensitivity, it is important to select a wavelength so that the signal change for a given particle size change is optimal. Because of the importance of refractive index for turbidometric detection of agglutination reactions, core materials are limited to those that produce acceptable signal changes for the desired assay sensitivity. For high concentrations of analyte (μg/ml range) the selection is not critical. However, for analytes in the nanogram 1 ml range, particles with a high refractive index are required. That is, core polymers with a high degree of aromatization and atomic weight substituents are preferred over aliphatic polymers, and high refractive index polymers are generally preferred over low refractive index polymers. The inner core of the polymer particle can be selected from a large group of materials with high refractive index. Preferred are materials that can be made by emulsion polymerization in such a way that the final particle size is controllable and substantially uniform. Typical polymers used for the inner core of polymer particles have a refractive index of 1.54 or higher (NaD line,
at 569 nm) and are listed in Table 1. Since the refractive index is a function of the wavelength, the scattering properties depend on the measurement wavelength. Generally the refractive index is greater at shorter wavelengths.

【table】

【table】

[Table] Doresin

Table: Not all of the above polymers can be used as the inner core for the particle reagents of the present invention. The reason is that there are other criteria that should be applied in the selection of the core monomeric material. For example, cellulose is not easily manufactured as uniform particle size spheres. Condensation polymers are also not useful. This is because the polymerization process does not produce spherical particles of the type that can be obtained by emulsion polymerization. Some thermoplastic polymers, such as poly(oxyethylene-oxyterephthalate) and some urea-formaldehyde type thermosets, are not suitable. Monomers of interest are those containing substituents which confer high refractive properties, such as halide, aromatic, heterocyclic, unsaturated or carbocyclic groups, as well as vinyl or allyl groups. Polymer particles useful for the preparation of the particulate reagents of the invention can be preferentially prepared by emulsion polymerization. Stepwise emulsion polymerization can lead to core/shell polymers close to the desired refractive index, no less than n _D =1.54. To obtain a polymer of the desired refractive index, it is preferred that the shell polymer does not exceed about 10 parts by weight of the polymer particles. A convenient method for particle size control of polymer particles is to first prepare a seed emulsion, the size of which can be controlled by the amount of surfactant used. After preparation of the seed emulsion, additional monomers and surfactants can be added at a controlled rate to increase the particle size in the seed emulsion. The outer shell polymer of the polymer particles can be made from a wide variety of ethylenically unsaturated monomers having functional groups capable of reacting with compounds of biological interest. Optionally, the outer shell can also contain other ethylenically unsaturated monomers. Attachment of the shell polymer to the core can be accomplished by grafting functional monomers onto residual ethylenically unsaturated groups in the core polymer or by polymerizing functional monomers around the core. can be used to generate adjacent shells. Preferred monomers include those containing epoxy groups, such as glycidyl methacrylate, glycidyl acrylate, vinyl glycidyl ether and methallyl glycidyl ether. Other functional groups include carboxyl, hydroxyl, amino and aldehyde. It is preferred to carry out the core monomer conversion to substantial completion so that the shell polymer is a homopolymer or copolymer of known composition rather than a copolymer of unknown composition. Conversions of greater than 98% can be achieved by raising the temperature of the core emulsion to about 95°C at the end of the polymerization. To further reduce the possibility of producing particles whose surface is a copolymer of unknown composition, the shell monomer can be added gradually rather than batchwise. In such methods, residual core monomer may be consumed during the early stages of shell polymer formation. If the monomers used are those containing epoxy groups, the shell polymer is preferably a homopolymer, but in practice it is preferred that up to 10 parts by weight of the inner core monomers of the outer shell be used. can be made to exist. In the case of shell monomers containing hydroxyl, amino, aldehyde or carboxylic acid groups, care must be taken to exclude the formation of water-soluble polymers. Thus, for example, acrolein or methacrylic acid alone cannot be used to generate homopolymer shell structures. However, they can be copolymerized with other monomers to produce water-insoluble polymer particles. It is also possible to modify the outer shell polymer by subsequent treatment with alternative chemical techniques to produce a covalently bondable surface. For example, epoxy groups can be hydrolyzed to produce diol compounds that can react with cyanogen bromide, which can act as a coupling agent for amine groups in proteins. Aldehydes can be reacted directly with amines to form Schiff bases which are then reduced to form covalent bonds. Alternatively, the aldehyde can be oxidized to produce an acid and the carbodiimide used in subsequent reaction with an amine to produce an amide bond. The outer shell is preferably a homopolymer. However, it may contain up to 10 parts, preferably up to 5 parts and even more preferably up to 2 parts of inner core monomer, based on the weight of the outer shell. These monomers may be residual monomers from the inner core polymerization. The particle reagents of the present invention can contain several different functional shell materials. Preferred ones contain epoxy groups that can be conveniently used to form covalent bonds with compounds of biological interest such as haptens, antibodies, proteins or hapten-protein conjugates. Such reactions result in the production of particulate reagents of the present invention. The production of the particle reagent can be carried out as follows. A hapten or proteinaceous material can be adsorbed onto the surface of a polymeric particle, for example, and then a functional group, such as an epoxide group, can be reacted with a complementary functional group on the hapten or proteinaceous material under appropriate PH conditions. Any unreacted material is then separated from the particle reagent. The reaction conditions are such that substantial cross-linking of particles should not occur. Substantial cross-linking results in the production of heterogeneous reagent particles and unexpected cloudy changes during subsequent immunoassays. There are two possible methods for making particle reagents containing compounds of biological interest covalently linked via proteinaceous materials. A compound of biological interest, such as a hapten, is first attached to the carrier protein and then attached to the polymer particles. Alternatively, the protein can be first attached to the polymer particles and then the hapten attached to the protein. The second approach has the advantage of using the same protein-particle reagents for the synthesis of particle reagents having various compounds of biological interest attached to them. Surface coating of the polymer particles with a hapten or protein material, i.e. the ratio of polymer particles to the compound of biological interest, depends on the reaction time, dilution of the compound of biological interest with an inert diluent, or additives to aid particle dispersion. It can be changed by Although complete coverage may produce the most rapid aggregation rate, less surface coverage may be important in increasing assay sensitivity. The resulting particle reagent can be further suspended in a substantially aqueous medium that may contain buffers, serum components, and surfactants to produce a monodisperse particle reagent for use in light scattering immunoassays. The present invention further relates to immunologically active stable particle reagents for use in sensitive light scattering immunoassays for the determination of compounds of biological interest. The targets of assay include a wide variety of substances for which immunologically corresponding reactive components in biological fluids, cells and tissue extracts may be produced. Compounds of biological interest include serum, plasma, saliva, urine or milk proteins, drugs, vitamins, hormones, enzymes, antibodies, polysaccharides,
These include bacteria, protozoa, fungi, viruses, cell and tissue antigens and other blood cell or blood fluid substances. Of particular interest are substances whose quantitative measurement is required for the evaluation of disease states and various drugs. This immunoassay can be performed in various ways depending on the type of analyte and the sensitivity required. For relatively high concentrations of analytes, such as certain serum proteins, appropriate antibody reagents can be used directly in particle-enhanced turbidimetric immunoprecipitation assays. The method of the invention provides increased detectivity, considerable savings in reagent costs compared to conventional immunoprecipitation techniques, and this allows the use of smaller patient sample volumes. Conversely, for the detection of circulating antibodies of interest, correspondingly reactive antigen or antibody particle reagents can be used directly in the assay. The inhibition immunoassay methods of the present invention also require bifunctional or polyfunctional agents in addition to the particle reagent. These are hereinafter referred to as flocculants for causing aggregation of the particle reagents. It is this aggregation that can be inhibited by compounds of biological interest. The aggregating agent can be a particle reagent based on an antibody to the compound of biological interest or a polymeric particle covalently bound to an antibody of the compound of biological interest as described above. These flocculants are used in cases where the particle reagents used in the method contain covalently bound compounds of biological interest. A flocculant can also be a multivalent conjugate of a compound of biological interest and a protein. Such conjugates are used when the particle reagent used in the method of the invention contains pores with covalently bound compounds of biological interest. Several different assay configurations can be used for the measurement of haptens. In one such configuration, antigenic particle reagents (hapten-particles or hapten-protein-particle reagents) can be produced and inhibition of the reaction of these particles with antibodies by compounds of biological interest can be performed. be measured. This reaction can be carried out by direct competitive reaction between the particles and the patient hapten for the antibody, or by a series of reactions between the hapten and the antibody followed by the addition of an antigenic particle reagent. Other assay configurations for haptens utilize antibody particle reagents whose aggregation with soluble multihapten-protein conjugates is inhibited by the analyte. Such assays can also be performed in an antagonistic or sequential manner. In still other assays, both antibody and antigen particle reagents of the same or different sizes can be present, and inhibition by the hapten can be performed in a competitive or sequential manner. The flocculation reaction that occurs in the method of the invention can be accelerated by the presence of a flocculation promoter. Such promoters may be polyethylene glycol or surfactants such as sodium dodecyl sulfate. This latter is particularly useful in digoxin assays using digoxin-HSA-particle reagents. The following examples illustrate the invention. Example 1 Preparation of polystyrene/polyglycidyl methacrylate shell-core polymer particles (a) A three round bottom flask equipped with a stirrer and a thermostatic heating mantle is used for the polymerization. The polymerization is carried out at 70°C in a nitrogen atmosphere. 6g “Gafac” RE610 (GAF
Anionic surfactant (available from the Company) and 2 g of potassium persulfate.
A seed emulsion is prepared by adding ml of styrene. After half an hour, 400ml of styrene, 4
ml of allyl methacrylate and 1.5 g of aerosol OT-100 (dioctyl sodium sulfosuccinate, available from American Cyanamid Company).
Start at a rate of ml/min. After the feed is complete, the emulsion is held at 70°C for 1 hour to ensure complete conversion of styrene. The final solids content was 15.9% and the particle size of the core polystyrene emulsion (determined by turbidity measurements at 546 nm) was
0.067 μm and the surface tension (measured with a tensiometer using the du Nouy ring method) is 65 dynes/cm ² . (b) 300 ml for the production of shell-core polymers;
A round bottom flask is used. Polymerization is carried out at 80°C under a nitrogen atmosphere. 200ml of the core polystyrene emulsion (Example 1a) was mixed with 50ml of water containing 0.2g of potassium persulfate and 0.2g of anhydrous potassium carbonate.
ml and then 3.9 ml of glycidyl methacrylate at a rate of 0.1 ml/min. After 45 minutes the mixture is allowed to cool. The final shell-core polymer has a particle size of 0.069 μm. Example 2 Preparation of polystyrene/polyglycidyl methacrylate shell-core polymer particles (a) A three round bottom flask equipped with a stirrer and a thermostatic heating mantle is used for the polymerization. The styrene is purified prior to polymerization by passing it through a column packed with basic alumina. Polymerization is carried out at 70°C under a slow nitrogen flow. Polymerization is initiated by adding 45 ml of styrene, 5 ml of ethylene glycol dimethacrylate, 50 ml of 30% aqueous sodium dodecyl sulfate solution and 2 g of potassium persulfate to 2 parts of deionized water. This mixture was polymerized at 70°C for 20 minutes. At this point, the particle size is 0.021 μm and its surface tension is 38.8 dynes/cm ² . This means that virtually all of the surfactant is used to stabilize the particles. The particles are then grown to a final size of 0.043 μm by gradually adding 400 ml of styrene and 10 g of "aerosol" OT-100 at a rate of 4 ml/min. Its final size is based on the initial particle size and the volume of added styrene.
It is predicted to be 0.044 μm. 590 ml of the seed emulsion prepared above are added to 1510 ml of deionized water containing 1.5 g of potassium persulfate and heated to 70°C.
When this mixture reaches 70°C, add 340 ml of styrene and 3.4 g of "Aerosol" OT-100
Add at a rate of ml/min. Once the feed is complete, the temperature is increased to 95° C. for 0.5 hour to ensure high monomer conversion. The conversion is 98.4% complete as determined by gas chromatography of the ether extract of the emulsion. The final core size is 0.070 μm (predicted value calculated from initial size and volume of added styrene is
0.069μm). (b) Add 200 ml of water containing 2 g of potassium carbonate and 2 g of potassium persulfate to the core polymer prepared in (a) above and adjust the reaction temperature to 80°C. 50 ml of glycidyl methacrylate are then added at a rate of 1.5 ml/min. Give a total of 45 minutes for shell polymerization. The final particle size is 0.71 μm and the glycidyl methacrylate conversion is 97.3% (using gas chromatography measurements as with styrene). Ultimately, styrene conversion is considered complete. The reason is that styrene is not detectable by chromatography. Example 3 Preparation of polyvinyl carbazole/polyglycidyl methacrylate shell-core polymer particles 300 ml with distillation head and mechanical stirrer
A round bottom flask is used. 200 ml of water containing 0.5 g potassium persulfate, 0.5 g trisodium phosphate hydrate and 1.5 g sodium dodecyl sulfate are heated to 70° C. in a nitrogen atmosphere.
A seed emulsion is produced by addition of 4.5 ml styrene and 0.5 ml ethylene glycol dimethacrylate. After 30 minutes, this seed emulsion has a grain size of 0.021 μm and a pH of 8.5. 20g vinylcarbazole and 1g in 10ml dichloromethane
The “aerosol” solution of OT-100 is then 0.1
Add at a rate of ml/min. Immediately after its addition, the dichloromethane is removed by distillation. After completion of the core emulsion, add 0.1 g potassium persulfate in 10 ml water followed by 2.5 ml glycidyl methacrylate.
Add at a feed rate of 0.1 ml/min. Allow 45 minutes for polymerization. The final particle size is 0.041 μm.
The final solids content is 10.5%. Example 4 Preparation of polystyrene/polyglycidyl methacrylate shell-core polymer particles (a) The method of Example 1(a) is used as follows. To water 2 containing 2 g of azobisisobutyramidine hydrochloride, add 50 ml of styrene and 2 g of cetyltrimethylammonium bromide and
Allow to polymerize for 30 minutes. Then a mixture of 200 ml of styrene and 2 ml of allyl methacrylate was added to
Add at a rate of mg/min. After the addition of 100 ml and again after the addition is complete, add 0.75 g of seltrimethylammonium bromide. The final solids content is 10.5% and the particle size is 0.106μm
It is. (b) 200 ml of core emulsion prepared in Example 4(a) above;
A mixture of 0.2 g of azobisisobutyramidine hydrochloride and 0.2 g of sodium acetate (anhydrous) dissolved in 10 ml of water was placed under a nitrogen atmosphere.
Heat to 70°C. After stabilizing the temperature, 3ml
of glycidyl methacrylate at a rate of 0.1 ml/min. Allow 45 minutes for polymerization. The final emulsion has a surface tension of 4.85 dynes/cm ² . Example 5 Preparation of gentamicin particle reagent and its use 50 mg of gentamicin sulfate in 5 ml of water are neutralized by adding barium hydroxide until no further precipitate forms. This barium sulfate precipitate is removed by centrifugation. This supernatant liquid was mixed with 5 ml of polystyrene/polyglycidyl methacrylate polymer particles (prepared in Example 4) and
0.1% "Schercozoline" in ml
A suspension of S (substituted imidazoline from stearic acid available from Shell Chemical Company) is added to the mixture adjusted to pH 8.5 with potassium hydroxide. The mixture is warmed to 75° C. for about 30 minutes, diluted with 200 ml of water and deionized using a mixed ion exchange resin bed. The resulting particle reagent 0.4mm to 20ml 0.1
% sodium dodecyl sulfate (SDS) plus 0.020M containing 0.3M NaCl and 0.1% SDS
Add 0.2 ml of phosphate buffer (PH7.43). 1.5 ml of this mixture by separately adding human serum, gentamicin, anti-gentamicin (rabbit antiserum, Kalestad Laboratories product), and a mixture of anti-gentamicin and gentamicin and measuring its turbidity. Test for immunological reactivity. The results, expressed in Table 2 as turbidity change rate, show that the particle reagent is immunologically active and can be used for the determination of gentamicin.

[Table] Example 6 Measurement of theophylline (a) Production of 8-(3-carboxypropyl)-1,3-dimethylxanthine 8-(3-dimethylxanthine), an 8-substituted theophylline derivative
-carboxypropyl)-1,3-dimethylxanthine
Pharmacology” Vol. 13, pp. 497-505 (1976)
It is synthesized as follows according to the published method. Glutaric anhydride (6.8 g) and 4,5-diamino-1,3-ditylpyrimidine-2,6-dione (6.2 g) were incubated in Dean Stark for 3 hours in 15 ml of N,N-dimethylaniline under nitrogen. Reflux using a trap. The mixture is allowed to cool, filtered and the solid product (3.6 g) is recrystallized twice from water. The purified derivative has a melting point of 254-255°C and an expected parent ion peak at m/e = 266 in the mass spectrum. (b) Attachment of theophylline derivative to polymer particles The 8-substituted theophylline derivative prepared in (a) above is attached directly to the polystyrene/polyglycidyl methacrylate polymer particles prepared in Example 1 as follows. 8-(3-carboxypropyl)-1,3-dimethylxanthine (50
mg) and 0.6 ml latex (17% solids)
0.015M sodium phosphate buffer (PH7.3,
50ml) for 1 hour. PH of the mixture
Adjust to 5.4 and heat for an additional hour. Then adjust the PH to 2.3 and heat for the last hour. This mixture was then centrifuged (80 min, 40,000×
g) and resuspended in an equal volume of 0.1% sodium dodecyl sulfate, recentrifuged, and again 0.1% sodium dodecyl sulfate.
Suspend in 0.015 sodium phosphate (PH78) containing 6000% polyethylene glycol (PEG). The reagents are then dialyzed against the same buffer. (c) Determination of theophylline This determination is carried out on an "aca" apparatus (DuPont product) at 37°C. 0.020ml of a sample containing an unknown amount of theophylline at 2.5% (W/V)
Add to 4.98ml of 0.15M phosphate buffer (PH7.8) containing PEG6000. Add 0.004 ml of rabbit anti-theophyllin antiserum (Kalestad Laboratories) and after a 3.5 minute incubation period, add the particle reagent prepared in (b) above containing 3% solids. 0.150
Start the reaction by adding ml. The turbidity increase due to particle aggregation is 29 seconds and 46 seconds after particle addition.
It is measured as the absorbance difference (rate of change) at 340 nm between seconds. Table 3 shows the data for the standard curve for the serum theophylline assay. Comparing the results obtained for an unknown sample to this curve gives the amount of theophylline present (theophylline standards are prepared by appropriately diluting a solution of theophylline of known concentration in water using human serum; The assay results provide data for the standard curve).

EXAMPLE 7 Conjugation of Human Serum Albumin to Polymeric Particles A method similar to that described herein was applied to IgG, various drugs, and drug conjugates such as digoxin-
HSA, theophylline-HSA, gentamicin-
It can be used to attach HSA, tobromycin-HSA and thyroxine-catalase conjugates to polymer particles. A solution containing 1 mg/ml HSA (0.3 M NaCl,
0.02M phosphate, PH 9.7) 6 ml of latex polymer particles (10% w/v) prepared in Example 1 are added dropwise to 1.2 with stirring and sonication.
The suspension is stirred, sonicated and heated (50° C., 1 hour). This suspension was then heated for 7 hours.
Dupont's "Sorvall" at 12500rpm
Centrifuge to remove all unbound HSA using a model RC-5B centrifuge and GS rotor.
resuspending the particle pellet in 500 ml of 0.1% sodium dodecyl sulfate (SDS) in the buffer;
Heat to 50°C for 1 hour and centrifuge again using the same conditions. 250ml of particle pellets 0.1%
Resuspend in SDS, sonicate and SS34
Centrifuge for 2 hours at 19500 rpm using a rotor.
Particle pellet 0.15M containing 0.1% HSA
Resuspend in 60 ml of 0.1 M Glycine Buffer (GBS Buffer) (PH 7.6) containing NaCl and then centrifuge for 80 minutes at 19500 rpm using an SM24 rotor. The particle pellet was finally resuspended in 60 ml of glycine-buffered saline/HSA solution to produce particles with proteins attached to their surface for possible subsequent covalent attachment of compounds of biological interest. Create a 1% (w/v) suspension of the reagents. It is stored at 4°C. Example 8 Determination of bovine serum albumin (a) Binding of BSA to polymer particles 20 mg of BSA (Sigma Chemical Co., Fraction V) was mixed with 0.3 M NaCl with 10 ml of 20 mM sodium phosphate (PH 9.7). 2.0mg/ml bovine serum albumin (BSA) by dissolving in
Produce a solution. Then 0.060 ml aliquots of latex polymer particles (Example 1) (14% w/v suspension,
0.069 μm diameter) to this BSA solution and sonicated for 1 hour at 50°C. Subsequent centrifugation and sonication conditions are as given in Example 7 with appropriate reductions depending on the volume used. (b) Assay of BSA Assay was performed using a 3.0 ml cuvette containing 2.0 ml of buffer [0.15 M sodium phosphate, pH 7.8, 4.5% (w/v) polyethylene glycol PEG6000] at 37°C. )
Performed on a 219 spectrophotometer. different level
0.050 ml of BSA-containing sample and 0.050 ml of antiserum (Miles Biochemicals product, antiserum)
BSA, undiluted) is added to the cuvette and allowed to incubate for 5 minutes before incorporating 0.050 ml of BSA-coated particles. When the BSA particle reagent prepared in (a) above is added, the degree of aggregation is monitored by tracking the change in absorbance at 340 nm. Table 4 shows that the rate of aggregation is inhibited by increasing the level of BSA in the sample.

[Table] Example 9 Measurement of haptoglobin (a) Binding of haptoglobin to polymer particles Polystyrene/polyglycidyl methacrylate polymer particle latex (17
%w/v suspension, 0.076 μm diameter, prepared in a similar manner to Example 1) in 20mM sodium phosphate (PH9.7)
Add to 5 ml of a solution containing 1.0 mg/ml human haptoglobin (Calbiochem Behring). "Branson" for 1 hour at 50℃
After sonication in a Model B-22-4 aqueous sonicator, the suspension is centrifuged at 19000 rpm in a DuPont Sorval Model RC-SB centrifuge. After 2 hours, the resulting particle pellet is resuspended in 5 ml of 0.1% (w/v) sodium dodecyl sulfate. After centrifugation for an additional 40 minutes, the resulting pellet was diluted with 5 ml of 0.2M glycine (PH
7.4)) and centrifuge again. Heat Systems Ultrasonics is used to completely disperse particles.
The final pellet is resuspended in 1 ml of 0.2M glycine by sonication for 5 minutes using a Model W-185-F sonicator. (b) Haptoglobin Assay The assay is performed on a Cary 219 spectrophotometer using a 3.0 ml quartz cuvette containing 2.0 ml of 0.150 M sodium phosphate buffer (PH 7.8) at 37°C. 0.050ml anti-human haptoglobin antiserum (Calbiochem-Behring product)
are incubated at 37° C. for 3 minutes with 0.040 ml of sample containing free haptoglobin in the buffer. After the incubation period, 0.050 ml of the haptoglobin particle reagent prepared in (a) above (0.10%
w/v particles) and follow the initial rate of absorbance change at 340 nm. Table 5 shows the measured rates as a function of haptoglobin concentration.

[Table] Example 10 Determination of gentamicin (a) Binding of gentamicin-human serum albumin conjugate to polymer particles 1.5 g of gentamicin sulfate (product of Sigma Chemical Company) in 50 ml of distilled water and
6.25 g N- in a solution of 1.5 g human serum albumin (no globulin, manufactured by Sigma Chemical Co.)
Ethyl-N'-(3-dimethylaminopropyl)
Add carbodiimide hydrochloride with stirring. Adjust the pH of the solution to 6.0 by adding 0.1N hydrochloric acid. This mixture was kept at room temperature for 18 hours and then
Dialyze against 15mM sodium phosphate buffer (PH7.8) at 4°C. The gentamicin-HSA conjugate is then carboxymethylated to prevent non-specific sedimentation of the particles. The buffered conjugate prepared above was diluted with 6M guanidine hydrochloride and 0.1M tris(hydroxymethyl).
Generate 500 ml of a solution containing aminomethane (PH 8.2). Gradually add 2.5 ml of β-methylcaptoethanol with stirring. this mixture
Hold at 37°C for 2 hours, then gradually increase while stirring.
Add 12.5 g of iodoacetic acid sodium salt.
Adjust its pH by adding 1N sodium hydroxide.
Readjust to 8.2. After 2 hours at 37°C, the product was dissolved in 0.30M sodium chloride and 20mM
Dialyze against phosphate buffer (PH7.8). All precipitated proteins were collected at 12500 rpm.
It can be removed by centrifugation for 20 minutes. The preparation of the gentamicin-HSA-particle reagent using the gentamicin-HSA conjugate is carried out by the method described in Example 7. (b) Assay for Gentamicin This assay was performed at 37°C using an “aca” apparatus.
It will be carried out in Sample containing gentamicin 0.010
ml to 4.99 ml of 0.15 M phosphate buffer (PH 7.8) containing 2.5% (w/v) polyethylene glycol (PEG) 6000. Antiserum from rabbit i.e. anti-gentamicin-BSA (product of Kalestad Laboratories) 0.015ml (1/2
50-1/500 final dilution). After culturing at 37°C for 35 minutes, the particle suspension prepared in (a) (1% w/v, 0.050 ml) is added to initiate the reaction. The turbidity increase is measured at 340 nm by the difference in absorbance between 29 and 46 seconds after particle addition. Table 6 shows the standard curve data for this assay. On the other hand, a comparison to the standard radioimmunoassay method is given in Table 7 (gentamicin standard is prepared by dissolving gentamicin sulfate in normal human serum and making appropriate dilutions). .

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[Table] Example 11 Determination of tobromycin The preparation of the tobromycin-HSA-particle reagent and the assay for tobramycin are performed using the method given for gentamicin (e.g.
10). Table 8 shows the inhibition of agglutination produced by serum tobromycin.

[Table] Example 12 Measurement of theophylline (a) Production of theophylline-HSA conjugate 8- required for coupling with protein
Substituted theophylline derivatives are synthesized by the method of Example 6(a). in 1.6 ml of N,N-dimethylformamide.
25 mg of 8-(3-carboxypropyl)-1,3
- To a solution of dimethylxanthine (prepared in Example 6) at 0 DEG C. are added 11 mg of N-hydroxysuccinimide and 21 mg of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was then stirred for 18 hours at 4°C and then added to human serum albumin (HSA,
120mg) solution. the resulting mixture
The pH is maintained at 9.0 by addition of 0.5N NaOH and the mixture is stored at 4°C for 18 hours. The resulting conjugate was dialyzed against deionized water,
It is then freeze-dried. Analysis of the final material is approx.
A molar ratio of theophylline to HSA of 20:1 is shown. (b) Production of theophylline-HSA-particle reagent A solution of theophylline-HSA (300 ml, 2 mg/
ml) in 0.02M bicarbonate buffer (PH9.7). Then 1.8 ml (15% solids) of the polymer particle latex prepared in Example 1 is slowly added with stirring. The mixture is heated to 70° C. for 1 hour and then centrifuged (80 minutes, 40,000×g) to remove all unbound conjugate. Resuspend the particles in an equal volume of 0.1% SDS and recentrifuge. Wash the particle reagent with 0.015M phosphate buffer (PH 7.8) by centrifuging and resuspending twice. Final suspension is 1mg/ml HSA
It is in the form of a 0.015M phosphate buffer (PH7.8) containing . Adjust this capacity to 1%
(w/v) to produce a solids particle concentration. (c) Theophylline assay This assay is carried out on an "aca" apparatus at 37°C. 0.030 ml of sample containing theophylline
ml 0.15M phosphate buffer (PH7.8)
and add this to 4.96 ml of the same phosphate buffer also containing 2.0% (w/v) PEG6000. Diluted with 0.040ml of 0.15M phosphate buffer (PH7.8) containing 0.1% HSA
Add 0.010 ml rabbit anti-theophylline antiserum (Kalestad Laboratories product) and after a 3.5 minute incubation period 0.050 ml of the particle reagent prepared in (b) above.
to start the reaction. Preparation of theophylline standards and turbidity measurements are performed as described in Example 6. Table 9 shows data for a standard curve for measuring serum theophylline using the drug-conjugate-particle reagent.

Table Example 13 Determination of thyroxine Preparation of thyroxine-catalase conjugate 5 g of L-thyroxine sodium salt (6.3 mmol) are suspended in 20 ml of 50% aqueous dioxane. 18.9 mmol (2.64 ml) of triethylamine was added to this suspension with stirring, followed by 1.71 g.
(6.93 mmol) of N-tert-butoxycarbonyl-2-phenylacetonitrile is added. After stirring for 2 hours at room temperature, 25 ml of H ₂ O and 33 ml of ethyl acetate are added and the aqueous and organic phases are separated. Extract this aqueous phase with 33 ml of ethyl acetate. Solid sodium citrate is added to the aqueous phase to lower the pH of the solution to 4.0. The resulting precipitate, N-(tert-butoxy)-L-thyroxine, is collected, dried under vacuum and stored at -20°C. 1.23 g (1.4 mmol) N-tert-butoxy)
-L-thyroxine and 0.16 g (1.4 mmol)
of N-hydroxysuccinimide is dissolved in 20 ml of dimethylformamide. 0.31 g (1.5 mmol) dicyclohexylcarbodiimide is added to this solution, which is then stirred for 18 hours at room temperature. The urea precipitated during the reaction is removed by filtration and the liquid containing the active ester is stored at -20°C. in 15ml of 0.15M sodium bicarbonate (PH8.1)
Dissolve 45 mg of catalase. 0.30 ml of active ester solution (150 mM) is added and the resulting suspension is stirred for 1 hour at 4°C. All unreacted esters are removed by centrifugation at 15000 rpm for 30 minutes, and the supernatant is then dialyzed against 10 mM potassium phosphate buffer (PH7). N
The -(tert-butoxy)-L-thyroxine-catalase conjugate is lyophilized and then stored at -20°C. (b) Preparation of thyroxine-catalase-particle reagent 30 mg of N-(tert-butoxy)-L-thyroxine-catalase conduit was prepared according to the method of Example 7, except that the reaction was carried out at 40°C in the absence of NaCl. A gate (10 ml of a 3.0 mg/ml solution in 10 mM potassium phosphate buffer (PH7)) is attached to the 0.069 μm particle size latex polymer particles prepared in Example 1. (c) Thyroxine assay Turbidity assay for thyroxine is carried out on an “aca” instrument. 0.15M phosphate buffer (PH7.8), 2.5% (w/v) PEG6000,
Add 0.10 ml to a 4.8 ml buffer containing 0.01% 1-anilino-8-naphthalenesulfonic acid (ANS) and 0.2% sodium salicylate.
Thyroxine samples with various concentrations of [0.2M
Prepared by dissolving L-thyroxine sodium salt in KOH and diluting in 0.15M phosphate buffer (PH 7.8) containing 0.1% HSA]. Add 0.050 ml of sheep antithyroxine antiserum [product of Abbott Laboratories, diluted to 1/5 with phosphate buffer (PH7) containing 0.1% HSA] to each aliquot;
After a 35 minute incubation period at 37°C, the turbidimetric reaction is initiated by adding 0.050 ml of 1% thyroxine-catalase particle reagent to the "aca" test pack. The rate of increase in turbidity at 340 nm is measured after 29 seconds and 46 seconds. Table 10 shows the data for the standard curve for this assay.

[Table] Example 14 Determination of digoxin (a) Preparation of digoxin-HSA conjugate Dissolve 2.40 g of digoxin in 144 ml of absolute alcohol. 144 ml of 0.1 M sodium periodate was added dropwise with stirring and the reaction was incubated at 30°C.
Continue for 25 minutes. At this point, add 3.264 ml of 0.1M glycerol to stop the reaction. After 5 minutes, the reaction mixture was dissolved in 144 ml of deionized water.
g of HSA [pH adjusted to 9.2 with 5% (w/v) potassium carbonate]. During the course of the reaction (30 min, 25
℃) Its PH using 5% (w/v) potassium carbonate
Keep it between 9.0 and 9.5. Added 1.272g of sodium borohydride and after 3 hours the pH was 3.65M
Adjust to 6.5 with HCl. This solution is then dialyzed against deionized water. After dialysis, the solution
Concentrate to 130ml using an Amicon concentrator using a PM10 filter. This solution was then lyophilized to release digoxin-
Generate HSA conjugates. Digoxin content analysis by sulfuric acid carbonization method and protein content analysis by absorbance at 280 nm per protein molecule.
Showing the ratio of digoxin molecules of 13.5. (b) Preparation of digoxin-HSA particle reagent Digoxin-HSA conjugate was prepared by the method given in Example 7 to 0.069 μm and
2 of 0.109μm (produced by the method of Example 1)
The species are bonded to differently sized latex polymer particles. (c) Digoxin assay This assay is carried out on an “aca” apparatus at 37°C. Standards (both in human serum and in a buffer (PH 7.5) or GBS buffer containing 0.1 M glycine, 1% NaCl, 0.01% sodium azide) were added in DMSO at 1 mg/ml digoxin aliquots and as appropriate. Manufactured by dilution. 100μm standard sample, 0.02M
Phosphate, 0.3M NaCl, 0.1% SDS, 0.01
4.9 ml of PH 7.5 buffer containing % sodium azide and then 50 μl of antiserum (rabbit antidigoxin from Capel Laboratories, 1
1/ml in GBS buffer containing mg/ml HSA.
50 dilution, 1/5000 final dilution). at 37℃
After a 3.5 minute incubation period, 0.050 ml of each of the two different digoxin-HSA particle reagents are added to initiate the reaction. Turbidity is measured at 340 nm as a rate (absorbance difference 29 and 46 seconds after start) or as an end point at various times after start. table
11 indicates the end point value in milli-absorbance units obtained at 340 nm using 0.109 μm particles. On the other hand, the table
12 shows velocity data using 0.069 μm particles. Data comparison shows that for the 0-50 ng concentration range the time required to achieve a given mA ranges from 15 minutes for use of smaller starting particles.
Indicates that the time will be shortened to 10 minutes. Table 13 shows that after starting with particle reagent (based on 0.06 μm polymer particles)
Data are shown for a standard curve where the rate of absorbance change was measured between 29 and 46 seconds. In comparison, Table 14 shows that even at lower antibody concentrations, there is some loss in sensitivity for particle reagents based on larger 0.109 μm polymer particles. For smaller particle reagents based on 0.069 μm particles, 500 ng/sample would result in 42% inhibition at a 1/1250 antibody dilution, whereas larger particle reagents would result in 42% inhibition at a 1/1667 antibody dilution. This results in a 22% reduction in speed. This example illustrates the improvement in assay sensitivity to digoxin when using smaller particles.

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【table】

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[Table] Example 15 Measurement of digoxin (a) Binding of antibodies to polymer particles The immunoglobulin G fraction (IgG) of a specific rabbit antiserum against digoxin-BSA conjugate (a product of Capel Laboratories) was
0.3M NaCl, 0.020M Sodium Phosphate and 1.0
in a 25ml buffer containing mg/ml HSA.
Dissolve to a final IgG concentration of 1.0 mg/ml. 1N
After adjusting the PH of the solution to 9.7 using NaOH,
0.069 μm diameter manufactured in the same manner as Example 1.
Gradually add 0.150 ml aliquots of 14% (w/v) polymer particle suspension. The resulting suspension was heated to 50°C and a "Branson" type B-
Gently sonicate for 1 hour using a 22-4 water bath sonicator. After sonication, the suspension is centrifuged for 2 hours, after which the resulting pellet is resuspended in 0.1% (w/v) sodium dodecyl sulfate and centrifuged again for 40 minutes. The pellet from the second centrifugation was washed with glycine-buffered saline (GBS, 0.1M glycine, 0.15M NaCl,
PH7.6) and centrifuge for an additional 40 minutes. The third centrifugation step produces a pellet of IgG-polymer particle reagent (flocculant). 1.0 this
Resuspend in 5 ml of GBS containing mg/ml HSA and 0.1% (w/v) sodium azide. This final suspension is used to disperse the particles before use.
20 minute heat systems ultrasonics
Sonicate with model W-185-F sonicator. All centrifugal stages are of the Dupont-Sobal type.
It is carried out using an RC-5B centrifuge using an SM-24 rotor at 19000 rpm. (b) Binding of digoxin-HSA to polymer particles Digoxin-HSA conjugate was 10mM
Digoxin in 50% ethanol for 30 min at 30 °C
Produced by reaction with 50mM sodium periodate. After stopping the reaction by adding 70mM glycerol, 2 volumes of this solution were mixed with 1 volume of 21.3 mg/ml adjusted to pH 10.1 with potassium carbonate.
Add to mlHSA. The digoxin-HSA conjugate solution is incubated for 1 hour at room temperature, after which sodium borohydride is added to the 50 mM level. After another 2 hours the pH is adjusted to 6.5 with 3N HCl. The resulting conjugate solution was adjusted to pH 7.8.
Dialyze against 5mM sodium phosphate. This digoxin-HSA conjugate solution is diluted with 20mM sodium phosphate, 0.3M NaCl buffer (PH9.7) to a final protein concentration of 2mg/ml. Diameter produced in a similar manner to Example 1
A 0.540 mM aliquot of a 14% (w/v) suspension of 0.069 μm polymer particle latex is added to 90 ml of conjugate solution and sonicated for 1 hour at 50°C. The conditions of sonication and subsequent centrifugation are the same as given in (a) above and this produces the digoxin-HSA-particle reagent. (c) Digoxin assay This assay contains 2.0ml of buffer [4.5%
Contains (w/v) polyethylene glycol 6000
37 using a 3.0 ml quartz cuvette containing 0.150 M sodium phosphate buffer with a pH of 7.8.
Performed in a Cary 219 spectrophotometer at °C.
(Anti-digoxin) IgG-particle reagent volume containing this buffer and 1.0 mg/ml HSA
Incubate for 5 minutes with 40μ of sample containing free digoxin in GBS. After incubation, digoxin-HSA
Add aliquots of particle reagent and monitor initial rate of absorbance change at 340 nm. Table 15 shows the concentration dependence of the absorbance change rate, and
16 Digoxin (40μ particle reagent and 50μ
shows inhibition of these rates by flocculants).

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Claims (1)

Claims: 1 having essentially (A) an inner core and an outer shell, where the inner core has a refractive index not less than 1.54 as measured at the wavelength of the sodium D line and the outer shell has ( 1) an ethylenically unsaturated monomer having a functional group capable of reacting with a compound of biological interest selected from the group consisting of epoxy, carboxy, amino, hydroxy and aldehyde; (2) optionally other ethylenically active monomers; unsaturated monomer,
and (3) a polymer of not more than 10 parts by weight of the inner core monomer of the outer shell, and the outer shell is prepared by polymerization in the presence of said inner core from 0.03 to 0.1. (B) a compound of biological interest or an antibody thereto to which said polymer particle is covalently bound, having a high refractive index; Particle reagent. 2. The particle reagent of claim 1, wherein said outer shell comprises no more than 5 parts by weight of said polymer particles. 3 consisting essentially of (A) an inner core and an outer shell, wherein the inner core has a refractive index not less than 1.54 as measured at the wavelength of the sodium D line and the outer shell comprises (1) epoxy, carboxy, an ethylenically unsaturated monomer having a functional group capable of reacting with a compound of biological interest selected from the group consisting of amino, hydroxy, and aldehyde; (2) optionally other ethylenically unsaturated monomers;
and (3) a polymer of not more than 10 parts by weight of the inner core monomer of the outer shell, and the outer shell is prepared by polymerization in the presence of said inner core from 0.03 to 0.1. (B) a compound of biological interest to which the polymer particles are covalently bound via a proteinaceous substance; A particle reagent having 4. For the determination of compounds of biological interest, (A)(1) essentially (a) has an inner core and an outer shell, the inner core being capable of being measured at the wavelength of the sodium D line; and the outer shell has (i) an ethylenic inorganic polymer having a functional group capable of reacting with a compound of biological interest selected from the group consisting of epoxy, carboxy, amino, hydroxy, and aldehyde; (ii) optionally other ethylenically unsaturated monomers; and (iii) not more than 10 parts by weight of the outer shell of the inner core monomer, and the outer shell is a polymer of the monomers described above. (b) an organism to which said polymer particles are covalently bound; and (b) an organism to which said polymer particles are covalently attached. a particle reagent with a high refractive index consisting of a compound of biological interest or an antibody thereto; (2) a liquid presumed to contain a compound of biological interest; and (3) a flocculant. and (B) spectroscopically measuring increased particle size resulting from aggregation. 5 The polymer particles are covalently bound to a compound of biological interest. A method according to claim 4. 6. The flocculant comprises (A) an antibody directed against a compound of biological interest; (B) essentially (1) has an inner core and an outer shell, where the inner core has a wavelength of the sodium D line; hand
an ethylenically unsaturated material having a refractive index not less than 1.54 and whose outer shell has (a) a functional group capable of reacting with a compound of biological interest selected from the group consisting of epoxy, carboxy, amino, hydroxy, and aldehyde; monomer, (b) optionally other ethylenically unsaturated monomers, and (c) not more than 10 parts by weight of the outer shell of the inner core monomer, and the outer shell is a polymer of said inner core monomer; (2) polymer particles having an approximate diameter range of 0.03 to 0.1 μm, which are produced by polymerization in the presence of a core; Claim 5 selected from the group consisting of a particle reagent having a high refractive index consisting of: an antibody against a compound of interest;
The method described in section. 7. The method of claim 4, wherein the monomeric particles are covalently linked to an antibody against a compound of biological interest. 8. The method of claim 7, wherein the flocculant is a multivalent conjugate of a compound of biological interest and a protein. 9. A method according to claim 4, wherein an aggregation promoter is also present during the culturing step (a). 10. The method of claim 9, wherein the aggregation promoter is selected from the group consisting of polyethylene glycol and sodium dodecyl sulfate. 11. The method of claim 5, wherein the polymer particles are covalently bound to a compound of biological interest via a proteinaceous substance. 12 essentially (A) having an inner core and an outer shell, wherein the inner core has a refractive index not less than 1.54 as measured at the wavelength of the sodium D line and the outer shell comprises (1) epoxy, carboxy, an ethylenically unsaturated monomer having a functional group capable of reacting with a compound of biological interest selected from the group consisting of hydroxy and aldehydes; (2) optionally other ethylenically unsaturated monomers;
and (3) a polymer of not more than 10 parts by weight of the inner core monomer of the outer shell, and the outer shell is prepared by polymerization in the presence of said inner core from 0.03 to 0.1. A particulate reagent having a high refractive index consisting of a polymeric particle having a diameter in the approximate micrometer range; and (B) an antigen of a compound of biological interest to which said polymeric particle is covalently attached. 13. Claim 12, wherein said outer shell comprises no more than 5 parts by weight of said polymer particles.
Particle reagent as described in Section. 14. A particle reagent according to claim 12, wherein the antigen of a compound of biological interest is bound to the polymer via a protein substance. 15. In measuring proteins, the following shall be used: ( ii) optionally other ethylenically unsaturated monomers, and (iii) a polymer of not more than 10 parts by weight of the inner core monomer, and which outer shell is in the presence of said inner core. (b) an antibody against a protein to which said polymer particles are covalently bound; (2) incubating a liquid presumed to contain a compound of biological interest; and (B) spectroscopically determining the increased particle size resulting from aggregation. A method that includes the steps of measuring. 16. The method of claim 15, wherein the protein is an antibody and the polymer particles are bound to the antibody or the antigen of the antibody.