KR20000063268A - Model Colloid for Diagnostic Usage, and Method for Producing Thereof - Google Patents

Abstract

PURPOSE: A model colloid for diagnostic reagent is provided, which shows even size of particle, and good surface character, so can be used in immunological diagnostic reagent field using Latex Agglutination Test; and which can control particle size and surface character of the model colloid in manufacturing process. CONSTITUTION: A process for the preparation of model colloid for diagnostic reagent comprises of: preparing Poly(St/AA) by emulsifying copolymerizing process without emulsifier at the first stage; pouring functional monomer AA with St at the second stage when reaches in an adequate electrification rate(the 2 stage shot-growth method), which can introduces much more functional monomers than existing method, and can control surface electric charge till -140microC/cm¬2(surface electric charge density); washing the particles using the ion exchange method and the serum replacement method, and calculating the surface electric charge density using the conductometric titration.

Description

Model Colloid for Diagnostic Reagent and Manufacturing Method Thereof {Model Colloid for Diagnostic Usage, and Method for Producing Thereof}

The present invention relates to a model colloid for diagnostic reagents and a method for manufacturing the same, and more particularly, to a monodisperse polymer latex colloid and a method for manufacturing the same.

Recently, monodisperse polymer latex, defined as a model colloid, has emerged as a promising application. The ideal model colloid is a monodisperse spherical particle that has been chemically modified with the desired amount of surface functionalities to have a very uniform particle size and a specialized surface.

Due to the characteristics of this model colloid, its application is quite wide. Monodisperse latex particles have been widely used as reference samples for electron microscopy, but the scope of application has been extended to reference samples for many other analytical instruments.

Synthetic techniques that can control various particle sizes and distributions are also a major factor in broadening the application of model colloids. In particular, the particle size of the model colloid has a great influence in the analysis of the mechanism of biological phenomenon such as phagocytosis. Characterized surfaces and surface charges of model colloidal particles are very important in studying the interaction between particles and proteins present in vivo.

In biomedical applications, model colloids have the advantage that they can be synthesized for a given application because they provide free particle size control, very uniform particle size, and a very wide solid / liquid interface.

One promising use of model colloids is in the field of immunological diagnostic reagents using the Latex Agglutination Test (LAT). In the diagnostic method using LAT, model colloids are used to identify and quantify antigens or antibodies present in trace amounts in blood serum, urine, and cerebrospinal fluid (CSF). The detection and quantification of certain antigens and antibodies is particularly important in the interpretation of human phenomena, since these antigen antibodies are important indicators of diseases such as meningitis and changes in body phenomena such as pregnancy. Antigen (Ag) is an antibody generator, forming a unique Ag-Ab complex by specific affinity binding with an antibody (Ab).

The antibody molecule has two divalent Ag-recognition sites capable of recognizing the antigen in the "Y" form, and each site binds to one specific site of the antigen molecule. Thus, if a large amount of antigen and antibody are present, the antigen-antibody complexes are cross-linked with each other by the divalent nature of the antibody molecule.

Model colloids can visualize and quantify antigens or antibodies by enhancing these antigen-antibody complex formation reactions.

To detect the antibody, a specific antigen is first coated on the surface of the model colloid particle. Since an antibody may bind with two antigenic sites which are the same at the same time, one antibody may bind between the same binding sites between adjacent colloidal particles, thereby allowing latex agglutination of the colloidal particles. A and B of the colloidal particle binding is illustrated in Figure 1 A and B, respectively.

When the antibody present in the sample is mixed with the latex particles coated with the antigen, as shown in FIG. 1B, the particles are agglutated to form a large mass and visualized. At this time, the degree of aggregation is proportional to the number of antibodies and particles involved in the reaction, so the degree of aggregation is measured by light scattering, turbidity, etc., and the detection and quantification of specific antibodies is the principle of the LAT (Latex Agglutination Test).

LAT is widely applied to the diagnosis of diseases such as rheumatoid disease, meningitis and men's diagnosis such as pregnancy.

However, model colloids, which play an important role in diagnostic reagents, are complicated to manufacture, and the size and surface properties of colloids vary greatly according to the properties of the monomers, which are difficult to commercialize due to their uneven adsorption. have.

Therefore, the present invention, to solve the disadvantages of the conventional model colloid as described above, to provide a new model colloid and a method for producing a colloid that is small in size and uniform, easy to manufacture, and excellent antibody binding to the particle surface. .

Furthermore, the present invention provides a simple particle distribution and an adsorption reaction of an antigen or an antibody which enables uniformity in the aggregation reaction of the antigen / antibody to be used for the LAT diagnostic reagent using the antigen-antibody specific binding. Provided is a model colloid with a surface functional group introduced therein and a method for preparing the same so that a stable adsorption reaction can occur.

In addition, the present invention provides a variety of modified model colloids surface-modified by introducing a functional monomer to the particle surface with a method of adsorbing the antibody by the hydrophobic attraction of the particles that provide a hydrophobic surface, such as polystyrene (polystyrene) To improve the binding of the particles for LAT with the protein antigen.

1 is a schematic representation of the latex flocculation process.

Figure 2 is an example of an apparatus for cleaning a model colloid according to the present invention.

3 is a titration curve of the model colloid according to the present invention.

4A and 4B are electron micrographs of model colloidal particles according to the present invention.

5a and 5b are graphs showing the aggregation rate in the antigen-antibody reaction using the model colloid according to the present invention.

The present invention for achieving the above object is one or two or more components selected from the group consisting of 100 to 150 parts by weight and MAA, NaSS, AA and AM selected from styrene or MMA based on monomers It relates to a model colloid for diagnostic reagents comprising 0.2 to 1.5 parts by weight.

The present invention also provides 100 to 150 parts by weight of one or two components selected from styrene or MMA based on monomers, 0.2 to 1.5 parts by weight of one or more components selected from the group consisting of MAA, NaSS, AA and AM, KPS as an initiator. It relates to a method for producing a model colloid for diagnostic reagents by mixing 0.01 to 0.05 parts by weight of the emulsion-free emulsion co-polymerization batchwise at 60 to 80 ℃.

The present invention also provides 100 to 150 parts by weight of one or two components selected from styrene or MMA based on monomers, 0.2 to 1.5 parts by weight of one or more components selected from the group consisting of MAA, NaSS, AA and AM, KPS as an initiator. 0.01 to 0.05 parts by weight of the emulsion-free emulsion co-polymerization in a batch at 70 ℃, and then swelling for 2 hours by adding MA as a functional monomer with St and the redox initiator is maintained at 40 to 50 ℃ for 24 hours It relates to a method for producing a model colloid for a diagnostic reagent to react.

In general, preparation methods for introducing functional monomers for controlling surface properties include batch-free emulsion-free emulsion copolymerization, two-step seed-batch-free emulsion-free copolymerization, and two-step shot-growth-free-free emulsion-free copolymerization. Taking two hydrophobic monomers as functional comonomers for styrene (St) surface charge control, methacrylic acid (MAA) is used as an example, and the outline of each method and the characteristics of the obtained model colloid are described as follows.

Batch-free emulsion emulsification copolymerization is a batch-free emulsion emulsion copolymerization by adding a large amount of St monomer and a small amount of MAA monomer (usually 99: 1). KPS (potassium persulpate) is used as an initiator and the polymerization temperature is 60 ~ 80 ℃. This method can not introduce more than 1% of the MAA, a functional monomer, it is difficult to control the surface charge, but has the advantage of obtaining a hydrophobic model colloid with a very small monodisperse particle size.

Two-stage seed-batch-free emulsion emulsification copolymerization is also known as two-stage swelling method. In the first stage, polystyrene (PS) seed is prepared by the emulsion-free emulsion polymerization method, and then MA is a functional monomer in two steps. To swell for 2 hours by adding with St, a redox initiator is added and then reacted for 24 hours while maintaining 50 ℃ in the reactor. This method can introduce more functional monomers to the surface than batch-free emulsifier emulsion copolymerization, but there is still a limit to the amount of functional monomers that can be introduced. On the other hand, in order to exclude the nucleation reaction in the two-step polymerization, monodisperse seed particles having a large particle size can be prepared using monodisperse seed particles.

In the two-step shot-growth emulsion-free emulsion copolymerization, Poly (St / MAA) is made by batch-free emulsion-free emulsion copolymerization in the first step. When the appropriate conversion ratio is reached, the functional monomer MAA is added together with St in a two-step reaction. This method can introduce much more functional monomers than the existing methods and can control the surface charge up to -140 μC / cm2 (surface charge density) in the range of 0.1-0.9 μm.

Unlike the general emulsion polymerization, the two-step shot-growth method has the advantage of being able to independently control the particle size and surface properties and introduce excessive surface charges into the monodisperse particles. In this method, the first step of polymerization is a low concentration of the functional monomer to polymerize the particles to give stability to the particles, to control the size of the particles, and the second step to control the ratio of the functional monomer and other monomers to instantaneously add the surface charge density To determine. The prepared particles are structurally stabilized under aqueous solution, uniform particle size and surface charge can be controlled, and also stable to electrolyte concentration, pH change, and freeze-thaw process. It can be called As such, the model colloid with adjustable particle size and surface charge can be applied to the development of particles for LAT using specific binding between antigens and antibodies.

In general, emulsion polymer composite particles prepared through two stages of continuous polymerization are called core / shell structures. Core / shell structure particle manufacturing in the first step to prepare a seed (seed) having a uniform particle size (sed) and then polymerized by adding a monomer to the seed prepared in step 2, it is possible to produce a uniform but large particle size In addition, the surface properties can also be controlled by adding functional monomers at the second stage.

The two-stage polymerization method can produce particles with desired components and forms. Recently, with the development of TEM technology, analysis and research on the form of particles have been actively conducted. An important factor in preparing the latex by seed emulsion polymerization is the interfacial tension between phases, and in addition to the core / shell structure, particles such as inverted core / shell forms can be obtained. The interfacial tension is measured using the drop volume method or the surface tensiometric method.

The size and surface properties of the particles produced through two-step emulsion polymerization are largely affected by two factors. Two factors are the thermodynamic and dynamic driving forces, which determine the particle size and surface properties. The polarity of the particle surface is controlled by the degree of hydrophilicity / hydrophobicity of the seed particles and the two-stage monomer, and may have different particle sizes and surface properties depending on the solid content or composition ratio of the monomer when the two-stage monomer is added.

The present invention will be described in more detail with reference to the following examples.

Example 1 Preparation of Model Colloid

The monomers were mixed in the ratio as shown in Table 1 below to prepare a model colloid according to the two-step shot-growth emulsifier-free emulsion copolymerization method.

In the first step, P (St / AA) was made by a batch-free emulsion emulsification copolymerization method, and when the proper conversion ratio was reached, the functional monomer AA was added together with St in a two-step reaction. By this method, much more functional monomers can be introduced than the existing methods, and the surface charges can be controlled up to -140 μC / cm2 (surface charge density) in the range of 0.1-0.9 μm. Unlike the general emulsion polymerization, the two-step shot-growth method has the advantage of being able to independently control the particle size and surface properties as well as introducing excessive surface charge into the monodisperse particles. In the first step of the polymerization, the functional monomer is polymerized at a low concentration to give stability to the particles, to control the size of the particles, and to the second step to adjust the ratio of the functional monomers and other monomers to instantaneously charge the surface charge. To determine the density. The prepared particles are structurally stabilized in aqueous solution, uniform particle size and surface charge can be controlled, and stable for electrolyte concentration, pH change, and freeze-thaw process. It could be called.

Through this process, the C.V. (Coefficient of variation) value was 0.01 or less, resulting in a fairly uniform particle. The maximum solids content of latex produced by the emulsion-free polymerization method is about 20% and C.V. In order to obtain uniform particles of <0.01, it was optimal to reduce the solid content to 15% or less.

In the case where the surface charge density of the prepared latex was about 30 μC / cm 2 or more, about 15% and as much as 33% of the functional monomers used formed the polymer electrolyte in the water phase or existed as a water-soluble polymer. Thus, the use of a large amount of functional monomer in the introduction of the functional monomer results in an increase in the maximum surface charge density, but as the amount remaining in the aqueous phase increases, the stability of the particles is destroyed and the formation of secondary particles or It was found that latex formed flocculation. Therefore, in the two-stage shot-growth method, up to about 25% of the two-stage functional monomers can be added compared to the base polymer, and if more than two, the particle size is not uniform (CV> 3%) and the stability of the latex itself is destroyed. Could know.

All the prepared particles were washed by ion exchange method and serum replacement method, and the surface charge density was calculated using conductometric titration. The ion exchange resin used for the ion exchange method was a mixed bed type of Dowex 1 or Dowex 50W. The purchased ion exchange resin was stirred twice each with 3 N NaOH and 3 N HCl, and then washed four times with tertiary distilled water. The washing apparatus is briefly shown in FIG.

Example 2 Properties of Model Colloids

Various characteristics of the model colloid prepared in Example 1 were analyzed.

(1) Measurement of surface charge density

Sulfonate-modified latex forms one break-through curve because both sulfate (SO ^-4 ) from the initiator (potassium persulfate) and sulfonate (SO ^-3 ) from sodium styrene sulfonate (NaSS) are strong acids. for a latex modified with a strong acid and a weak acid (COO ^-) it has two points of the titration. In this case, finding the appropriate point is not easy. In the present invention, Metrohm's automatic titrator was used and titration was performed under nitrogen atmosphere.

Amide-modified latex (AML) is hydrolyzed with COOH, so about 3.5g of NaOH is added to 100g of latex solids, and then stirred at 97 ° C for 24 hours and then cooled to room temperature. Then, after the ion exchange presented above, potentiometric titration was performed.

Potentiometric titration calculated break-through titration by changing pH. Titration examples of strong acids and latexes containing weak acids are shown in FIGS. 3A and B, respectively. There is only one equivalent point as an example of the strong acid-strong base titration shown in FIG. 3A. In the case of CML or AML shown in FIG. 3B, since it is titrated with a strong base, there are two equivalent points in the neutralization reaction of the strong acid generated from the initiator and the weak acid generated from the carboxyl group.

Surface charge density was calculated from the following equation.

In the above formula, dp is the density of particles, Np is the equivalent of functional groups on the surface per 1g of latex solids, ΔV is the volume of the titrant, Wp is the mass of the solids of the latex.

Table 2 shows the surface charge characteristics of the model colloid.

(2) stability of latex

Samples stored after ion exchange showed a slowing down when the particle size was greater than 0.6 μm. In addition, even when all of the electrolyte and the polymer electrolyte was removed through the serum replacement cell, the stability was slightly decreased. In this case, it was possible to increase the stability by using a nonionic emulsifier such as Triton-X405. Even without using an emulsifier, it was re-dispersed again with a light shaking.

(3) change of surface charge density over time

Sulfonate-modified latex did not hydrolyze over time and maintained the initial surface charge density. However, in the case of CML and AML, the surface charge density decreased with time or temperature. The results are shown in Table 3 below.

(4) SEM analysis of model colloid

Each model colloid prepared in Example 1 was taken with an electron microscope and shown in FIGS. 4A to 4J (same as the sample sequence shown in Table 1), respectively.

As can be seen from the figure, it can be seen that the model colloid according to the present invention has a very uniform spherical shape with a particle diameter of 0.05 to 0.5 μm depending on the type.

Application Example 1: Applied to Immunoassay

Protein adsorption and desorption analysis studies were conducted using the model colloid prepared above.

Bovine serum albumin (BSA, Sigma Chemical Co., A-7906) was used as an antigen, and aniti-bovine serum albumin (anti-BSA, Sigma Chemical Co., B-7276) was used as an antibody. This antibody is characterized by polyclonal. It was dissolved in ultrapure water before use. As a model colloid, a model colloid prepared using 99% styrene and 0.2% NaSS monomer was used. In addition, a carboxylated model colloid was also used for comparative experiments. As mentioned above, the latex was washed by ion exchange resin and serum replacement. The model colloids used are shown in Table 4 below.

Coupling of BSA and model colloids was performed by physical adsorption. Acetate buffer of pH 4.5 was used and weak acidity was maintained in consideration of the pI of BSA. The amount of adsorbed BSA was measured using the turbidity method. The remaining BSA was washed with the same buffer solution in a stirred cell. BSA is adsorbed to the model colloid by two modes. In general, BSA is reversibly adsorbed by the side-on mode and the maximum adsorption amount in this mode is about 2.5 to 3.0 mg / m2. As the concentration of BSA increases, the structure of the BSA is irreversibly modified, which is called end-on mode.

Latex agglutination test was performed while stirring the BSA-coupled microsphere with the solution in which the antibody was dissolved. The surface area of the particles was 8.33 × 104 m 2 / mL and the degree of aggregation was measured using a spectro-photometer (UV-160A, Shimadzu, Japan). The measurement wavelength was 600 nm and it was confirmed that no other impurities were absorbed at this wavelength.

(1) Analysis of absorbance changes and non-specific agglutination (NSA)

5 and 6 show the agglutination ratios for the amount of antibody. Cohesion rate is defined as follows.

A.R, represents the aggregation rate, A0 represents the absorbance before the anti-BSA addition, and ΔA represents the change in absorbance through aggregation. As shown in FIG. 5A, the absorbance is increased in proportion to anti-BSA at lower concentrations at low carboxylated microspheres. This means that there is no antigen on the surface to form the antigen-antibody complex. The initial slope shows the maximum at a surface concentration of BSA of 1.0 mg / m2. By initial slope is meant the sensitivity of the sensitization ability to immunodiagnostic reagents.

As shown in FIG. 5B, low sulfonated microspheres show a larger initial slope than low carboxylated microspheres, especially when the surface concentration of BSA is 1.0 mg / m 2, no flat areas of carboxylated microspheres appear. Only a short transition region appears, again increasing above 20 μg / ml. This re-increasing phenomenon indicates that aggregation in this region is caused by non-specific agglutination (NSA) rather than by antigen-antibody response. This means that it does not occur in carboxylated microspheres, but is involved in the flocculation process on sulfonanted surfaces.

In FIG. 6A, the transition region is clearly seen as the surface sulfonate group increases. This means that the NSA increases with increasing surface sulfonate groups. Therefore, it can be concluded that the re-increasing phenomenon in FIGS. 5B and 6A is due to NSA.

(2) sensitization sensitivity to various microspheres

As shown in Table 4, comparing the sensitivity to sensitization is meaningless because the diameters of carboxylated and sulfonated microspheres are different. This is because larger particles' turbidity generally appears larger than smaller ones and the change in turbidity is greater. Therefore, low sulfonated microspheres (221 nm) are more sensitive than low carboxylated microspheres (195 nm). However, at low surface charge concentrations and high sulfonated microspheres (221 nm and 258 nm, respectively), microspheres with high surface charge concentrations exhibit lower sensitivity despite their large size. This is due to NSA because it reduces the general antigen-antibody response. Therefore, NSA is considered to decrease sensitivity.

Since the model colloid according to the present invention has a uniform particle size and excellent surface properties, it can be applied to the field of immunological diagnostic reagents using latex agglutination test. In addition, the present invention provides an optimal manufacturing method that can control the model colloid, which is the core technology for the development of LAT diagnostic reagent, to the desired particle size and surface properties.

By the present invention

(1) surface-characterized monodisperse particles, which provide a model colloid that acts as a stable substrate upon the adsorption of antigens or antibodies,

(2) can be surface modified with a carboxyl group or the like to allow covalent bonding on the surface with an antigen or antibody,

(3) uniform particle size can impart uniformity in antigen / antibody coating and aggregation reaction,

(4) When the antigen is bound to the model colloid, the binding with the antibody is performed at a very high speed by rapid brownian motion, and thus quantification can be performed within a short time.

Claims (3)

100 to 150 parts by weight of one or two components selected from styrene or MMA based on the monomer and 0.2 to 1.5 parts by weight of one or two or more components selected from the group consisting of MAA, NaSS, AA and AM Model colloid for diagnostic reagents. 100 to 150 parts by weight of one or two components selected from styrene or MMA based on monomers, 0.2 to 1.5 parts by weight of one or more components selected from the group consisting of MAA, NaSS, AA and AM, 0.01 to 0.05 parts by weight of KPS as initiator A method of producing a model colloid for a diagnostic reagent, characterized in that the mixture is mixed with the emulsion to batch emulsion-free emulsifier at 60 ~ 80 ℃. After the copolymerization according to claim 2, the functional monomer MA is added together with St to swell for 2 hours, and after the addition of a redox initiator, the diagnostic reagent model is reacted for 24 hours while maintaining 40 to 50 ° C. Method for producing a colloid.