JPS5850646B2 - 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 extraction-based diagnostic reagents.

Latex reagents for serological diagnosis generally have a particle size of 0.0.
A test reagent in which antigens or antibodies are sensitized (adhered) to latex particles of 0.05 to 1 μm and dispersed in a weakly alkaline buffer, and the test reagent 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.

Collodion particles, activated carbon, kaolin, bentonite, human and animal red blood cells, etc. have been known as particles that sensitize antigens and antibodies since before the development of latex reagents, but at present, their stability during storage is Polystyrene particles are widely used because of their excellent properties and the fact that the particles themselves have no antigenicity.

Polystyrene latex for simple chemical 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. is stabilized.

However, according to conventionally used emulsifiers,
Because the obtained latex contains free emulsifiers, it may already aggregate when antigens or antibodies are sensitized, and even if it can be effectively sensitized, it does not contain the antigen or antibodies to be detected. Contact with negative serum may cause an agglutination reaction.

Causing such a non-specific agglutination reaction is fatal for a test reagent.

Of course, the free emulsifier contained in the latex can also be removed from the latex by, for example, an ion exchange method or a dialysis method.

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 is severely damaged and cannot be put to practical use as a latex reagent.

The present invention was made to solve the above-mentioned problems in latex for serological diagnostic reagents, and it uses a mono(meth)acrylic acid ester of (poly)alkylene oxide that has both radical polymerizability and emulsifying action. By emulsion copolymerizing styrene and water in the presence of ultrafine silicic anhydride, the resulting latex contains virtually no free emulsifier, and therefore has excellent serology without causing nonspecific agglutination reactions. The purpose of the present invention is to provide a method for producing latex for diagnostic reagents.

The method for producing latex for serological diagnostic reagents of the present invention includes:
Styrene and the general formula (where R1 and R2 are each independently H or CH3
, R3 means H or an alkyl group, and n is 1 to
Represents the number 100.

) is copolymerized in water with a water-soluble radical polymerization initiator in the presence of ultrafine particulate silicic acid anhydride.

The above (meth)acrylates are specifically mono(meth)acrylates such as polyethylene glycol, polypropylene glycol, and block copolymers of ethylene oxide and propylene oxide, and the molecular chains forming the (meth)acrylates The molecular chain end opposite to the end may remain a hydroxyl group, or may form a lower alkyl ether such as methyl ethyl or propyl.

Preferably n is 20 to 80, and one type or a mixture of two or more types is used.

These can be obtained as commercial products.

Specific examples include the following.

Such (meth)acrylates are 0 compared to styrene.
．． It is used in an amount of 1 to 70% by weight, preferably 1 to 50% by weight, particularly preferably 3 to 30% by weight.

Ultrafine silicic anhydride means silicic anhydride with a diameter of 5 to 50 mμ, such as Aerosil (Japan Aerosil ■)
is preferably used.

Further, a colloidal solution of ultrafine silicic anhydride, such as Snowtex (Nissan Chemical Industry ■), is also preferably used.

The amount of ultrafine silicic anhydride used is 0.0% relative to styrene.
0.01 to 10% by weight, preferably 0.02 to 5% by weight.

In addition, as the water-soluble radical polymerization initiator used in the present invention, persulfates such as potassium persulfate, ammonium persulfate, and sodium persulfate, 2,2'-azobis(2-
amidinopropane) mineral acid salts, azo compounds such as azobiscyanopropane acid and its alkali metal salts and ammonium salts, redox catalysts such as tartaric acid peroxide, Rongalitto peroxide, ascorbic acid peroxide, etc. are mentioned, but persulfates are preferably used.

The amount of these polymerization initiators used is usually 0 relative to styrene.
．． 01-1% by weight.

The method of the present invention involves copolymerizing styrene and (meth)acrylate in water in the presence of ultrafine silicic anhydride as described above, and the polymerization reaction is usually carried out under heating while stirring. The temperature range is 50-100℃,
Preferably it is 65-85°C.

The time required for the polymerization reaction depends on the styrene concentration, the type and amount of polymerization initiator used, and the reaction temperature, but is usually 5 to 50 hours.

By the method of the present invention as described above, the average particle size is 0.05.
A latex of ~2μ is obtained, and one feature of the method of the present invention is that a monodisperse latex with very uniform particle size is obtained.

The variation in particle size is 0.05 or less, expressed as the coefficient of variation in particle size obtained by dividing the standard deviation of particle size by the average particle size.

The method for sensitizing the latex particles thus obtained with an antigen or antibody is not particularly limited, and conventionally known methods can be employed as appropriate.

For example, latex particles and antigens or antibodies may be mixed at a pH of about 7-8.
．． 6 in an appropriate aqueous solvent such as buffer solution, physiological saline, water, etc.
Contact is carried out at a temperature of about 20-37°C for a suitable time.

At this time, stir or shake if necessary.

The latex reagent thus obtained is further washed with an aqueous solvent or centrifuged to remove antigens or antibodies not adsorbed to the latex particles, if necessary.
It is redispersed in a buffer solution etc. to make a latex reagent.

In the latex reagent according to the present invention, in which the latex is sensitized with an antigen or antibody as described above, the (meth)acrylate copolymerized with styrene functions as an emulsifier during the reaction and after polymerization, keeping the latex stable. In addition, since this latex does not substantially contain free emulsifier, unlike conventional latex reagents containing free emulsifier, non-specific agglutination reaction does not occur.

Furthermore, according to the present invention, as shown in the following Examples and Comparative Examples, the emulsion copolymerization of styrene and (meth)acrylate is carried out in the presence of ultrafine silicic anhydride; Although not obvious, the sensitivity of the resulting latex reagents is improved several times.

Example 1 Styrene 65.9 Formula % Formula % 4.5 g of methacrylate with n of about 30, 0.05 g of potassium persulfate, 30% ultrafine particulate silicic anhydride colloid solution Snowtex 0.05.9 and ion exchange After charging 450 g of water into a reaction vessel and purging the inside of the vessel with nitrogen gas, the mixture was stirred at a temperature of 70°C for 30 hours to carry out an emulsion copolymerization reaction.

The average particle size of the latex thus obtained was 0.54μ,
The coefficient of variation in particle size was 0.04.

This latex was dispersed in glycine buffer at pH 8.5, and 1 volume of this dispersion was mixed with 1 volume of human gamma globulin solution diluted to 0.1% with glycine buffer.
The latex particles were kept at a temperature of A latex dispersion was obtained.

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, instead of the rheumatoid factor-containing serum, a rheumatoid factor-free serum was diluted 20 times and tested in the same manner using the above latex reagent, and no latex agglutination was observed.

The particle size coefficient of variation was 0.48μ and 0.036.

This latex was treated in the same manner as in Example 1 to obtain a latex reagent, and the results of the same tests are shown in the table.

In addition, when the same test as in Example 1 was conducted using serum that does not contain rheumatoid factor, no agglutination was observed.

In Example 3, X is about 30, 10 g, potassium persulfate 0.0! l, ultrafine particulate Aerosil silicic acid anhydride 0.1g
and 450 g of ion-exchanged water were charged into a reaction container, and after purging the inside of the container with nitrogen gas, the reaction was carried out at 80° C. for 23 hours.

The average particle size of the latex thus obtained was 0.40μ,
The coefficient of variation in particle size was 0.031.

A latex reagent was prepared from this latex in exactly the same manner as in Example 1, and the results of testing are shown in the table.

Furthermore, as in Example 1, no agglutination was observed for serum containing no rheumatoid factor.

Example 4 The polystyrene latex obtained in Example 1 was dispersed in phosphate buffer (PBS) at pH 7.4 to a solid content of 2%.

Separately, an anti-hepatitis B virus surface antigen (HBs antigen) monospecific antibody produced by a guinea pig (purified product by affinity chromatography by passing the solution twice through a column of antigen immobilized on Sepharose 4B) was added to the same PBS as above. One volume of this solution was mixed with one volume of the latex and incubated at a temperature of 37° C. for 2 hours to sensitize the latex particles with the antibody.

This sensitized latex was centrifuged at 15,000 rpm for 15 minutes to remove unadsorbed antibodies and separate the sensitized latex.

The antibody titer in the supernatant was measured by a passive hemagglutination assay (PHA method), and it was found that at least 99.5% of the antibody was adsorbed to the latex particles.

The above precipitated sensitized latex was further centrifuged at 15,000 rpm, the supernatant was discarded, and the precipitated sensitized latex was centrifuged at pH 7.
A latex reagent was prepared by redispersing the mixture in PBS.

Using this latex reagent, human serum containing HBs antigen at various concentrations was tested and the results shown in Table 2 were obtained.

Next, sera from 1000 normal people with HBs antigen of 0.4 n fl/rrtl or less (EI for detecting HBs antigen) were used.
When we conducted an agglutination test using the above-mentioned latex reagent for Kit A (confirmed using Reverseia J (Yamanouchi Pharmaceutical ■)), there was only 1 false positive out of 1000 samples, and it was found that it contained HBs antibodies. There were no false positives among the sera from 100 people.

Comparative example: 65 g of styrene, 20 g of methacrylate, the same as in Example 2.
g1 potassium persulfate 0.0! l and ion exchange water 450
g was charged into a reaction vessel, the inside of the reaction vessel was replaced with nitrogen gas, and then emulsion polymerization was carried out at a temperature of 60° C. for 40 hours.

The average particle size of the latex thus obtained was 0.53 μm, and the coefficient of variation of particle size was 0.05.

This latex was sensitized in the same manner as in Example 1 to prepare a latex reagent, and the same tests were conducted to obtain the results shown in Table 1.

The sensitivity is clearly lower than that of the latex reagent shown in Examples.

However, when a serum containing no rheumatoid factor was tested in the same manner as in Example 1, no agglutination was observed.

Claims (1)

[Claims] 1. Styrene and the general formula (where R1 and R2 are each independently H or CH3
, R3 means H or an alkyl group, and n is 1 to
The number is 100. ) A method for producing a latex for serological diagnostic reagents, which comprises copolymerizing a (meth)acrylate represented by () in water using a water-soluble radical polymerization initiator in the presence of ultrafine particulate silicic anhydride. 2 Ultra-fine silicic anhydride from 0.01 to styrene
2. A method for producing a latex for use as an extraction diagnostic reagent according to claim 1, characterized in that the latex is present in an amount of 10% by weight. 3. The method for producing a latex for extraction-based diagnostic reagents according to claim 1 or 2, wherein the water-soluble radical polymerization initiator is a persulfate.