JPS5837325B2 - Method for producing latex for diagnostic reagents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing latex for use in immunoserological diagnostic reagents.

Immunoserological diagnostic methods, in which polystyrene latex is sensitized with antigens or antibodies and used to detect the corresponding antibodies or antigens in serum as a latex agglutination reaction, are clinically popular due to their simplicity and rapidity. In the field of testing, it has been widely applied to detect many types of antigens or antibodies.

The polystyrene latex used for this purpose generally has a particle size of 0.05 to 1 micron, and preferably has a narrow particle size distribution and uniform particle size.

It is said that such a latex can be produced using a commonly known emulsion polymerization method.

The method is, for example, by mixing one or more of anionic, nonionic, or cationic emulsifiers in water with a styrene monomer and a water-soluble radical polymerization initiator, preferably. It is maintained at an appropriate temperature for an appropriate amount of time in an atmosphere excluding oxygen.

In the polystyrene latex thus obtained, the form in which the emulsifier exists, which contributes to its stability, is important.

Generally, part of the emulsifier used during polymerization is adsorbed on the surface of polystyrene latex particles, and the other part exists in the latex in a free state. There is an adsorption/desorption equilibrium on the particle surface.

In polystyrene latex produced by conventional methods, emulsifiers are essential for forming a stable latex.

However, free emulsifiers have an adverse effect on the latex agglutination reaction caused by the aforementioned antigens or antibodies.

That is, in order to produce a latex reagent, it is first necessary to sensitize polystyrene latex with an antigen or antibody as described above, but if latex containing free emulsifier is used, aggregation may already occur at this stage. .

Next, using latex sensitized with antigen or antibody,
When detecting an antibody or antigen corresponding to this antigen or antibody by a latex agglutination reaction, the sensitized latex will agglutinate if it comes into contact with the serum (positive serum) containing the antibody or antigen to be detected, and such antibodies or antigens will be detected. It is essential that the sensitized latex does not agglutinate even if it comes into contact with serum that does not contain antigens (negative serum).

However, in the case of sensitized latex containing free emulsifier, it often aggregates even when it comes into contact with negative serum, resulting in a non-specific agglutination reaction.

Of course, the free emulsifier contained in the latex can be removed using techniques such as ion exchange or dialysis.

However, when the free emulsifier is removed from the latex, the stability of the latex becomes extremely poor because the latex is stabilized by the establishment of adsorption/desorption equilibrium with the free emulsifier as described above. In reality, it becomes unusable.

As mentioned above, as a latex for use in immunoserological diagnostic reagents, polystyrene latex produced by the usual emulsion polymerization method has a major practical drawback in that it contains free emulsifiers.

The present invention was made as a result of intensive research aimed at providing a latex for use as an immunoserological diagnostic reagent free from the above-mentioned drawbacks. The present invention relates to a method for producing a latex for diagnostic reagents, which comprises copolymerizing in water using a water-soluble radical polymerization initiator in the presence of silicic acid.

As the styrene sulfonic acid salt used in the present invention,
Examples include sodium styrene sulfonate, potassium styrene sulfonate, lithium styrene sulfonate, ammonium styrene sulfonate, and the proportion of these compounds to the styrene monomer is preferably 10% by weight, more preferably 0.0001 to 10% by weight. %, particularly preferably o. ooi% to 5% by weight.

The ultrafine particles of silicic anhydride in the present invention have a particle size of 5
Silicic anhydride made into ultrafine particles of about 50 mμ is used, and for example, Aerosil (trade name, manufactured by Nippon Aerosil ■) is preferably used.

A colloidal solution prepared by dispersing the ultrafine silicic anhydride in water, such as Snowtex (trade name, manufactured by Nissan Chemical Industries, Ltd.), is also preferably used.

The amount of ultrafine silicic anhydride used is preferably 0.01 to 10% by weight based on styrene;
．． It is preferably 0.05 to 5% by weight, particularly 0.08 to 2% by weight.

In addition, when used as the above colloidal solution, the silicic anhydride content is 35% by weight or less, and the sodium I-IJium oxide content is 0.
．． 6% by weight or less and a viscosity of 10 centipoise or less.
It is preferable to use one having H8 to H10.

In addition, as the water-soluble radical initiator in the present invention,
Potassium persulfate, ammonium persulfate, sodium persulfate
Persulfates such as IJum, 2-2'-azobis(2-amidinopropane) mineral acid salts, azo compounds such as azobiscyanobuarelic acid and its alkali metal salts and ammonium salts, tartaric acid monoxide, longa Examples include redox initiators such as Litto peroxide and ascorbic acid monoperoxide, and persulfates are preferably used.

The ratio of these polymerization initiators to the heptamer is 0.
A range of 0.01 to 1% by weight is preferred.

To carry out polymerization for latex production according to the method of the present invention, styrene monomer, a salt of styrene sulfonic acid, ultrafine particles of silicic anhydride, or ultrafine particles of silicic anhydride are dispersed in water in a reactor filled with water. The colloidal solution and initiator may be added and heated while stirring, and the polymerization temperature at that time is usually 50 to 100°C, preferably 65°C.
It is preferable that the temperature is in the range of 35°C to 35°C.

The time required for the polymerization reaction varies depending on conditions such as the monomer concentration, the concentration of ultrafine particles of silicic anhydride or a colloidal solution in which ultrafine particles of silicic anhydride are dispersed in water, and the concentration of the initiator, but it is usually 5 to 50 hours. range.

Thus, by the method of the present invention, particles with an average particle size of 0.05 to 2 microns and a variation in particle size of 0.05 or less expressed as a coefficient of variation (standard deviation of particle size/average particle size) can be greatly reduced. A well-organized monodisperse latex can be obtained.

The latex obtained by the method of the present invention is extremely stable and has a very uniform particle size, and unlike the latex obtained by the conventional method, the emulsifier does not exist in a free state. It has excellent performance as an immunoserological diagnostic reagent without causing so-called non-specific agglutination reactions.

In the present invention, since ultrafine particles of silicic anhydride are used together with styrene and styrene styphonate, the resulting latex is extremely stable as described above, and can be prepared as an immunoserological diagnostic reagent for diagnosis. When used for this purpose, it can exhibit extremely high sensitivity.

Next, examples of the present invention will be described.

Example 1 A colloidal solution in which 91 g of styrene monomer, 0.66 g of sodium styrene sulfonate, 0.4 g of magnesium sulfate heptahydrate, and 0.09 g of ultrafine particles of silicic anhydride (average particle size of about 30 μ) were dispersed in water. 0.21, potassium persulfate O. :l, 440g of ion-exchanged water was charged into a reaction container,
The container was purged with nitrogen gas and copolymerization was carried out at a reaction temperature of 70°C for 24 hours.

The average particle size of the latex thus obtained was 0.6
1 micron, and the variation in particle size is expressed as a coefficient of variation of 0.
It was 04.

The latex thus obtained was dispersed in a glycine buffer with a pH of 8.5 to 1 volume of a latex dispersion, and 1 volume of the latex was mixed with a glycine buffer of 0.0. After mixing 1 volume of human gamma glopurin solution diluted to 1% and keeping at 30°C for 15 minutes**,
6. Unadsorbed human gamma puline was removed by centrifugation using OOOXG, and the precipitated latex particles were redispersed in glycine buffer to obtain a uniform sensitized latex dispersion.

One drop of this and one drop of serum containing rheumatoid factor diluted to various ratios with glycine buffer were mixed on a glass plate.
The strength of the agglutination reaction was observed by tilting the glass plate gently back and forth and left and right for a minute, and the results shown in the following table were obtained.

Furthermore, when the same test was performed using rheumatoid factor-free serum diluted 20 times with glycine buffer instead of rheumatoid factor-containing serum, no agglutination was observed.

As is clear from these results, the immunoserological diagnostic reagent prepared using the latex obtained by the method of the present invention has high sensitivity and does not cause non-specific agglutination reactions.

Comparative Example: 91 g of styrene monomer, 2 g of nonionic emulsifier (Daikomi Kogyo Seiyaku Emulgit 49), 0.3 potassium persulfate.
440 g of L ion-exchanged water was charged into a reaction vessel, the vessel was purged with nitrogen gas, and polymerization was carried out at a reaction temperature of 70°C for 24 hours.

The average particle size of the obtained latex was 0.48 microns, and the variation in particle size was 0.15 expressed as a coefficient of variation.

Using this latex, an immunoserological diagnostic reagent was prepared in exactly the same manner as in the example, and the strength of the agglutination reaction with serum containing rheumatoid factor was observed, and the results shown in the following table were obtained.

Furthermore, when 1000 cases of normal human serum were tested using this latex reagent, 96 cases of non-specific agglutination were observed.

As is clear from these results, the immunoserological diagnostic reagent prepared using the latex obtained by the method of the comparative example has a strong nonspecific agglutination reaction and low sensitivity.

Claims (1)

[Claims] 1. A method for producing a latex for diagnostic reagents, which comprises copolymerizing styrene and a salt of styrene sulfonic acid in water using a water-soluble radical polymerization initiator in the presence of ultrafine particles of silicic anhydride.