JPS5915321B2 - Method for producing latex for serological diagnostic reagents - Google Patents

Description

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

Latex reagents for serological diagnosis generally have a particle diameter of 0.
．． A test reagent in which antigens or antibodies are sensitized (adhered) to latex particles of 005 to 1μ and dispersed in a weakly alkaline buffer solution, which is reacted with the corresponding antibodies or antigens in serum to cause a latex agglutination reaction. These specific antibodies or antigens are detected as follows.

Currently, it is widely used in the field of clinical tests such as rheumatoid factor detection and pregnancy diagnosis due to its simplicity and speed. Particles that sensitize antigens and antibodies include:
Collodion particles, even before latex reagents were developed. Activated carbon, kaolin, bentonite, human and animal red blood cells, etc. are known, but polystyrene particles are currently widely used because of their excellent stability during storage and the fact that the particles themselves are not antigenic. There is. Polystyrene latex for serological diagnostic reagents has conventionally been most commonly produced by emulsion polymerization of styrene in the presence of one or more types of anionic, nonionic, and cationic emulsifiers.

The emulsifier has the greatest effect on the stability of this latex reagent. In the latex obtained by emulsion polymerization, some of the emulsifier used is adsorbed on the polystyrene particles, and some is in a free state, and an adsorption-desorption equilibrium of the emulsifier is established on the surface of the polystyrene particles. This equilibrium stabilizes the latex.・0 However, according to conventionally used emulsifiers, since the obtained latex contains free emulsifiers, it may already aggregate when sensitizing antigens or antibodies, and furthermore, it may not be possible to sensitize effectively. Even if it is possible, it may come into contact with negative serum that does not contain the antigen or antibody to be detected, causing an agglutination reaction. Causing such a non-specific agglutination reaction is fatal for test reagents. Of course,
The free emulsifier contained in latex can be removed by, for example, ion exchange or dialysis. It can also be removed from 0.

However, as mentioned above, the stability of latex is due to the adsorption-desorption equilibrium between the free emulsifier and the emulsifier adsorbed on the latex particle surface, so removing the free emulsifier from the latex reduces the stability of the latex. It was severely damaged and could not be put to practical use as a latex reagent. The present invention was made in order to solve the above-mentioned problems in the latex for serological diagnostic reagents, and it is an excellent latex that does not substantially contain free emulsifiers and does not cause non-specific agglutination reactions. The purpose of the present invention is to provide a method for producing latex for serological diagnostic reagents.

The method for producing latex for serological diagnostic reagents of the present invention includes:
This method is characterized in that styrene is polymerized in water using persulfate as a polymerization initiator in the absence of an emulsifier, and then heated under alkaline conditions.

Polystyrene obtained by polymerizing styrene in water using persulfate as a polymerization initiator in the absence of an emulsifier is known to have sulfate groups (SO42-) at both ends of the molecular chain (Polymer Chemistry, Vol. Volume 22, No. 244, Page 481 (196
Furthermore, due to their hydrophilic nature, these sulfate groups are distributed on the surface of polystyrene particles, and as a result, polystyrene particles are relatively stable due to electrical mutual repulsion despite the absence of an emulsifier. Forms a fine latex. However, the sulfate group at the end of the molecular chain is relatively unstable.
That is, the terminal end of a molecule having a sulfate group tends to form a carboxyl group via a hydroxyl group by hydrolysis. In this case, if the hydrolysis of the sulfuric acid group remains at the stage of forming a hydroxyl group as in Tsukasa 1), the latex becomes unstable because the hydroxyl group at the end of the molecular chain is difficult to dissociate.

\Kai▼●l-. Therefore, in the present invention, by heating polystyrene having a sulfuric acid group at the end of the molecular chain under alkaline conditions, following the hydrolysis of formula), a carboxyl group is introduced as shown in the following formula [). Oh, and ^
Substantially all of the sulfated sulfate groups are hydrolyzed into dissociable carboxyl groups, thus stabilizing the latex.

The persulfate used in the present invention is not particularly limited, but
Potassium persulfate, sodium persulfate, ammonium persulfate, and the like are preferably used, and the ratio of these to styrene is usually 0.01 to 5% by weight.

Polymerization of styrene may be carried out according to the emulsion polymerization method, and is stirred at a temperature of 50 to 100°C, preferably 60 to 90°C, for 5 to 50 hours under a nitrogen stream. The hydrolysis of the polystyrene latex obtained in this way involves the use of alkaline substances such as hydroxides, oxides, carbonates, and bicarbonates of alkali metals or alkaline earth metals in the latex. Dissolve an appropriate amount of sodium, potassium hydroxide, sodium carbonate, etc. and adjust its pH to 7-14, preferably 8-14.
12, heated to a temperature of 50 to 100°C, preferably 60 to 90°C, and stirred. The atmosphere should be airy. The polystyrene latex thus obtained usually has an average particle size in the range of 0.05 to 2 microns, is stable, and has extremely small variations in particle size.

That is, the coefficient of variation obtained by dividing the standard deviation of the particle size by the average particle size is 0.05 or less, and is a so-called monodisperse latex. The method of sensitizing the latex particles with an antigen or antibody is not particularly limited, and any conventionally known method can be employed as appropriate.

For example, latex particles and antigens or antibodies may be mixed at a pH of about 7 to
8.6 in an appropriate aqueous solvent such as a buffer solution, physiological saline, water, etc. at a temperature of about 20 to 37° C. for an appropriate time. On this occasion,
Stir or shake if necessary. If necessary, the sensitized latex reagent thus obtained is further washed with an aqueous solvent or centrifuged to remove antigens and antibodies that are not adsorbed to the latex particles, and then redispersed in a buffer solution, etc. Use as a latex reagent. The latex reagent obtained by sensitizing the latex obtained by the method of the present invention with an antigen or antibody as described above can be used as a latex reagent containing a conventional emulsifier, as clearly shown in the following examples. It has no non-specific agglutination reactions, which are often seen in reagents, and is highly sensitive, making it possible to make far more accurate diagnoses than conventional latex reagents.

Example 457 styrene, 0.044 y of potassium persulfate, and 450 f of ion-exchanged water were charged into a reaction vessel, and after purging the inside of the vessel with nitrogen gas, polymerization was carried out at a temperature of 70° C. for 30 hours.

After the reaction is completed, the atmosphere inside the reaction vessel is replaced with air, and the pH is
8.5 and stirred at 70°C for 24 hours. The average particle size of the latex thus obtained was 0.38μ, and the coefficient of variation in particle size was 0.03. This latex was dispersed in a glycine buffer with a pH of 8.5, and 1 volume of this dispersion was mixed with 1 volume of a human gamma globulin solution diluted to 0.1% with a glycine buffer.
After keeping it for 5 minutes, it was centrifuged at 26,000G to remove unadsorbed human gamma globulin to the latex particles.Then, the precipitated latex particles were again dispersed in the glycine buffer to form a uniform sensitized latex dispersion. I got it.

One drop of this latex reagent and one drop of human serum containing rheumatoid factor diluted to various ratios with glycine buffer were mixed on a glass plate, and the glass plate was gently tilted back and forth for 3 minutes to determine the strength of the agglutination reaction. The results shown in the table below were obtained. Furthermore, when a rheumatoid factor-free serum was diluted 20-fold in place of the rheumatoid factor-containing serum and tested in the same manner using the above latex reagent, no latex aggregation was observed.

Comparative example Styrene 91y, nonionic emulsifier (Daiichi Kogyo Seiyaku Co., Ltd.)
Emulsion 49) 27, 0.3 t of potassium persulfate, and 440 t of ion-exchanged water were charged into a reaction vessel, and after purging the inside of the reaction vessel with nitrogen gas, emulsion polymerization was carried out at a temperature of 70° C. for 24 hours.

The average particle size of the latex thus obtained was 0.48μ, and the coefficient of variation in particle size was 0.15. This latex was sensitized in the same manner as in the example to prepare a latex reagent, and the same test was conducted to obtain the results shown in the table.

Claims (1)

[Claims] 1. A method for producing a latex for serological diagnostic reagents, which comprises polymerizing styrene in water using persulfate as a polymerization initiator in the absence of an emulsifier, followed by heating in alkaline conditions.