JPS6116944B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a latex reagent for detecting antigens or antibodies. Recently, latex reagents have come into general use to detect trace amounts of biological components such as hormones and proteins in body fluids. As the latex, synthetic resin latex, especially one with a particle size of 0.1 to 1 micron, is generally used. In these latex reagents, antibodies and antigens corresponding to the antigens and antibodies to be measured are adsorbed on the surface of the latex, and a specific reaction occurs between antigens and antibodies.
The antibody reaction causes latex aggregation. However, simply adsorbing antibodies (or antigens) to the surface of latex inevitably causes non-specific agglutination due to factors other than the specific reaction of antigens and antibodies. cannot be obtained. This is because the body fluid to be measured contains various proteins, etc., and these components non-specifically adsorb to the latex and cause aggregation. In order to eliminate this drawback, various efforts have been made and reported so far, but the representative method is a method of treatment using an inactive protein that does not participate in antigen-antibody reactions. For example, Japanese Patent Publication No. 43-12741 describes an immunochemical measurement reagent in which an antigen or antibody is adsorbed onto a fine solid carrier. Latex reagents characterized by being coated with antigens have been proposed. In addition, Japanese Patent Publication No. 11407/1983 proposed a method for producing a latex reagent, which is characterized by adsorbing antibodies onto the surface of a fine solid carrier and then treating this with a solution of an inactive protein at a concentration of 0.1% or less. There is. Although there are differences in the operating procedures in these two inventions, the basic method is to treat with an inactive protein that does not participate in the immunochemical reaction of antigens and antibodies. Examples of this inactive protein include albumins such as bovine serum albumin, ovalbumin, and lactalbumin. However, latex reagents treated with albumin cause significant non-specific reactions with proteins in normal rabbit serum and normal guinea pig serum, resulting in agglutination. Furthermore, the measurement sensitivity is low due to latex self-aggregation due to the properties of albumin. Naturally, the sensitivity is low because non-specific reactions occur with human gamma globulin, human albumin, and human fibrinogen, and it is often necessary to perform an inhibition test (a type of confirmation test) by adding an antibody in advance. . Regarding the measurement sensitivity, that is, the detection limit concentration, using a latex reagent for detecting HBs antigen (hepatitis B virus surface antigen) as an example, a latex reagent manufactured by a normal albumin treatment method has a concentration of 10 μg (10 _-5 g/ cc) degree or higher
Currently, only HBs antigen can be detected, and something even higher is desired. In addition to visually identifying and diagnosing the agglutination phenomenon of latex reagents based on the antigen-antibody reaction, it is also possible to use visible light, near-infrared light, laser, etc. to detect the reduction in turbidity when a sample is applied to latex reagents. A method is known in which the antigen or antibody to be measured is measured using a standard curve prepared in advance using known concentrations of antigen or antibody.
For example, JP-A-53-24015 and JP-A-54-108693 disclose measurement methods using near-infrared light, but even in such optical measurement methods, the aforementioned non-specific reaction There are similar problems such as aggregation and low sensitivity. In view of the above-mentioned drawbacks of conventional latex reagents, the present invention has been made with the aim of providing a latex reagent that is free from non-specific aggregation and has excellent sensitivity. but (Here, R ₁ is H or CH ₃ , R ₂ is H or CH ₃ , and x, y, and z are each positive integers, and one of the following relationships holds true between them: 1≦x≦100, y=0, x=0 1≦y≦100, x=0, z=0 1≦x≦50, 1≦y≦50, 1≦z≦50, x+y+z≦100) The antibody is sensitized to a latex obtained by copolymerizing the same compound as described above in water using a water-soluble radical polymerization initiator in the absence of an emulsifier. A method for producing a latex reagent, which comprises dispersing it in a liquid containing serum containing serum and treating it by separating it from the liquid. Examples of the compound represented by the above general formula include acrylic or methacrylic esters of polyethylene oxide, acrylic or methacrylic esters of polypropylene oxide, and acrylic or methacrylic esters of block copolymers of ethylene oxide and propylene oxide. Specifically, etc. are given as suitable examples. These compounds are copolymerization components with styrene, and at the same time they also function as emulsifiers in commonly known emulsion polymerization methods, and the ratio of use to styrene is 0.1.
from 1 to 70% by weight, preferably from 1 to 50% by weight
% by weight, more preferably 3 to 30% by weight. In the above relational expression of x, y, z, x, y,
When z>100, the copolymerizability of styrene and these compounds decreases, making it impossible to obtain a stable latex with well-uniformed particle sizes. In addition, as the water-soluble radical polymerization initiator in the present invention, persulfates such as potassium persulfate, ammonium persulfate, and sodium persulfate, 2'-2 azobis(2-amidinopropane) mineral acid salt, and azobiscyanopoule are used. Azo compounds such as phosphoric acid and its alkali metal salts and ammonium salts, tartaric acid peroxide, Rongarit peroxide, ascorbic acid
Examples include redox initiators such as peroxides,
Persulfates are preferably used. The proportion of these polymerization initiators to the total monomers is preferably in the range of 0.01 to 1% by weight. To carry out copolymerization for producing latex by the method of the present invention, at least one of the compounds represented by the above general formula and the above initiator are added to styrene in a reactor filled with water, and the mixture is stirred. All you need to do is heat, and the polymerization reaction temperature at that time is usually 50 to 50℃.
The temperature is preferably 100°C, preferably in the range of 60 to 85°C. The time required for the polymerization reaction varies depending on conditions such as monomer composition, monomer concentration, and initiator concentration, but is usually in the range of 5 to 50 hours. Note that in the present invention, no emulsifier that is commonly used in the production of latex is used. Further, in the present invention, it is possible to coexist ultrafine particles of silicic anhydride during the above-mentioned copolymerization, and in this case, more favorable results are obtained due to the uniform dispersibility of the produced latex. Therefore, as the ultrafine particles of silicic anhydride, silicic anhydride formed into ultrafine particles with a particle size of about 5 to 50 mμ is used, and for example, Aerosil (trade name, manufactured by Nippon Aerosil Co., Ltd.) is preferably used. . In addition, a colloidal solution in which the ultrafine particulate silicic anhydride is dispersed in water, such as Snowtex (trade name, manufactured by Nissan Chemical Industries, Ltd.)
is also suitably used. The amount of ultrafine silicic anhydride used is 0.01 to 10% by weight based on styrene.
It is preferable to use a proportion of 0.05 to 5% by weight, especially
It is preferably 0.08 to 2% by weight. When used as the above-mentioned colloidal solution, it is preferable to use one having a silicic anhydride content of 35% by weight or less, a sodium oxide content of 0.6% by weight or less, and a viscosity of 10 centipoise or less and a pH of 8 to 10. In the present invention, by carrying out the copolymerization as described above, the average particle size is 0.05 to 2 microns, and the variation in particle size is 0.05 or less expressed as the coefficient of variation (standard deviation of particle size/average particle size). A monodisperse latex with very uniform particle size can be obtained.
The latex obtained by the above method is extremely stable and has a very uniform particle size, and unlike conventionally obtained latexes, the emulsifier does not exist in a free state, so it is highly immunizable. As a serological diagnostic reagent, it does not cause so-called non-specific agglutination reactions. Next, in the present invention, the latex obtained as described above is sensitized with an antibody, but the method of sensitizing this antibody is not particularly limited, and conventionally known methods can be adopted as appropriate. .
For example, latex particles and antibodies have a pH of about 7 to 8.6.
Contact is carried out 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. At this time, stir or shake if necessary. The sensitized latex thus obtained is further washed with an aqueous solvent or centrifuged, if necessary, to remove antibodies (or antigens) that are not adsorbed to the latex particles. In the present invention, the sensitized latex prepared above is treated by dispersing it in a liquid containing serum containing the same antibodies as above and then separating it from the liquid. is added to a buffer solution containing antibody-containing serum, i.e., antiserum, at an optimal concentration, suspended and stirred for a short time to treat the serum, and after removing excess serum, add preferably 0.5 to 3 weight of antiserum to the buffer solution. % concentration. The concentration of the serum in the buffer solution containing the serum is 0.1 to 20% by weight, and therefore the concentration of the antibody is 1 to 1000 μg/
It is preferable that it is about cc. Further, the temperature and time during the sensitization may be the same as those for antibody sensitization. As mentioned above, the latex reagent produced according to the present invention is different from the latex reagent produced by treating antibody-sensitized latex with an inert protein that does not participate in the antibody-antigen reaction based on the conventional method. It was treated with serum containing sensitized antibodies, and surprisingly, this treatment successfully removed the nonspecific agglutination reaction of the latex reagent and dramatically increased the measurement sensitivity. As shown in the Examples described below, it has become possible to make accurate diagnoses even for extremely low concentrations of antigens by measuring agglutination reactions with the naked eye or with an optical measuring device. For example, HBs antigen (hepatitis B virus surface antigen)
Taking latex reagents for detection as an example, latex reagents produced using conventional albumin treatment methods are
Whereas only HBs antigen of approximately 10 μg (10 _-5 g/cc) or more can be detected, the latex reagent produced based on the present invention can detect antigen amounts of approximately 10 ng (10 _-8 g/cc). In this case, it is actually 1000 times more sensitive. As described above, according to the present invention, it is possible to easily obtain a latex reagent that is free from non-specific agglutination and has extremely high sensitivity. Examples of the present invention will be described below. Example 1 Styrene monomer 65g 4.5g of the compound represented by, potassium persulfate 0.05
g and 450 g of ion-exchanged water were charged into a reaction vessel, the vessel was purged with nitrogen gas, and copolymerization was carried out at a reaction temperature of 70°C for 30 hours. The average particle size of the latex thus obtained was 0.59 microns, and the variation in particle size was 0.05 expressed as a coefficient of variation. One volume of this latex was dispersed in phosphate buffer at pH 7.4 with a solid content of 2%, and the solution was passed twice through a column containing HBs monospecific antibody produced by guinea pigs (antigen immobilized on Sepharose 4B). (purified product by affinity chromatography)
was mixed with 1 volume of the same solution dissolved in phosphate buffer at a concentration of 40 μg/cc and incubated at 37° C. for 2 hours to bind the antibody to this latex.
Next, this sensitized latex was centrifuged at 15,000 rpm for 15 minutes to remove the adsorbed antibodies. The antibody titer in this supernatant was measured by PHA (passive hemagglutination), and at least 99.5% of the antibodies were adsorbed to this latex. Next, guinea pig antiserum from guinea pigs immunized with HBs antigen and anti-human protein antibodies which have been absorbed in a column was added to the precipitated latex at a concentration of 0.1 to 0.1%.
The sensitized latex was well dispersed by adding 5% and 1 volume of phosphate buffer, and stirred at 37°C for 10 minutes. Anti-HBs in phosphate buffer containing this guinea pig antiserum.
The antibody is contained at approximately 1-50 μg/cc. Then, centrifuge at 12,000 rpm and discard the supernatant.
The precipitated treated sensitized latex was redispersed in a phosphate buffer solution of pH 7 to complete the preparation of the latex reagent. Using the latex reagent thus prepared, the agglutination strength of human serum containing various concentrations of HBs antigen was measured, and the results shown in the following table were obtained.

[Table] Next, Riverseia (HBs antigen detection EIA kit,
Serum HBs antigen was 0.4n using Yamanouchi Pharmaceutical)
A similar test was conducted on 1,000 normal human serum samples known to be below g/cc. There was only one false positive out of 1000 samples. Additionally, there were no false positives among the 100 people who contained HBs antibodies. Comparative Example 1 The same test was conducted using an anti-HBs antibody sensitized latex prepared in exactly the same manner as in Example 1, except that the treatment with guinea pig serum containing HBs antibody in Example 1 was replaced with treatment with normal guinea pigs. I got the result.

[Table] In addition, there were 9 false positives for 1000 normal human serum samples similar to those in the example, and 4 false positives for the reaction with 100 human serum containing antibodies. That is, although the non-specific reaction is not so great in this method, the detection sensitivity is about 1/1000 times lower than in the example. Example 2 The latex obtained in Example 1 was sensitized with an antibody to alpha-fetoprotein produced by domestic rabbits (a monospecific antibody purified by affinity chromatography), and alpha-fetoprotein in human serum was sensitized to the latex obtained in Example 1. The agglutination reaction with fetoprotein was investigated. The preparation method of the latex reagent is the same as in Example 1,
In this example, treatment was performed with a phosphate buffer containing 1% rabbit antiserum. The results are shown in the table below.

[Table] In addition, there were only 2 false positives among 1000 normal human serum samples. Comparative Example 2 As in Example 2, the latex obtained in Example 1 was sensitized with alpha-fetoprotein antibody and washed with a phosphate buffer containing 1% bovine serum albumin to prepare a latex reagent. . The agglutination reaction with alpha-fetoprotein in human serum was as shown in the table below.

[Table] It is clear that this comparative example has a detection sensitivity of only 1/1000 of that of Example 2. Also,
There were as many as 130 false positives out of 1,000 serum samples from normal people. That is, in the albumin treatment method, the reaction of non-specific aggregation is extremely significant. Example 3 Take 0.5 cc of the anti-HBs latex reagent prepared in the same manner as in Example 1 into a small test tube, add 0.5 cc of phosphate buffer, and then add 0.5 cc of the anti-HBs latex reagent prepared in the same manner as in Example 1, and then add 0.5 cc of the phosphate buffer solution to the solution at the concentration shown in the table below.
Add 1 c.c. of HBs antigen solution and mix by shaking for 20 seconds, then put into an acrylic resin cell equipped with a rotor (light path length 1 cm) and immediately stir at a speed of 200 revolutions per minute. Changes in absorbance over time were recorded.
The measurement wavelength used was 950 nm. Next, on this record of absorbance changes over time, a straight line is drawn along the approximate straight line as soon as possible after the start of recording, and the slope of the straight line is calculated as shown in Table 5 below. This is the number in the "Speed" column, which shows the change in absorbance in ABS/min.

[Table] Comparative Example 3 Antibodies prepared in exactly the same manner as in Example 3 except that the treatment with guinea pig serum containing HBs antibody in Example 3 was changed to treatment with normal guinea pig serum.
When the antigen-antibody reaction was optically measured using the HBs latex reagent, only an antigen amount of 5 μg/cc or more could be detected, and non-specific agglutination occurred. Example 4 (1) Preparation of anti-α-phetoprotein (α-FP) antibody sensitized latex (anti-α-FP latex) reagent, injecting rabbit-produced α into the same latex obtained in Example 1. - Sensitized with FP antibody (monospecific antibody purified by affinity chromatography). The latex reagent preparation method was the same as in Example 1, and the latex reagent was treated with a phosphate buffer containing 1% rabbit antiserum. (2) Optical measurement of latex agglutination reaction Example 3 except that the anti-α-FP latex reagent prepared in (1) above was used and visible light of 650 nm was used.
The results shown in Table 6 were obtained in exactly the same manner.

【table】

[Table] Comparative Example 4 Anti-α-
When the antigen-antibody reaction was optically measured using the FP Latex reagent, only an antigen amount of 7 μg/cc or more could be detected, and non-specific agglutination occurred.

Claims (1)

[Claims] 1. Styrene and the general formula are (Here, R ₁ is H or CH ₃ , R ₂ is H or CH ₃ , and x, y, and z are each positive integers, and one of the following relationships holds true between them: 1≦x≦100, y=0, x=0 1≦y≦100, x=0, z=0 1≦x≦50, 1≦y≦50, 1≦z≦50, x+y+z≦100) The antibody is sensitized to a latex obtained by copolymerizing the same compound as described above in water using a water-soluble radical polymerization initiator in the absence of an emulsifier. 1. A method for producing a latex reagent, which comprises dispersing the serum in a liquid containing serum, and then separating the latex reagent from the liquid. 2. The method for producing a latex reagent according to item 1, wherein the copolymerization is carried out in the coexistence of ultrafine particles of silicic anhydride.