JPS6116941B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a latex reagent for detecting an antigen. 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.
Diagnosis using these latex reagents is performed by adsorbing the antigen to be measured, an antibody corresponding to the antibody, and the antigen on the latex surface, and causing latex agglutination using a specific antigen-antibody reaction. be exposed. However, simply adsorbing antibodies (or antigens) to the surface of latex inevitably causes nonspecific agglutination caused by factors other than the specific reaction of antigens and antibodies.
A practical latex reagent 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, causing aggregation. In order to eliminate this drawback, various efforts have been made and reported so far, and 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 operating procedures in these inventions, the basic method is treatment with an inactive protein that does not participate in the immunochemical reaction of antigens and antibodies. Examples of inert proteins 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, taking 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/cc (10 ^-5 g/cc). It is only possible to detect HBs antigens at levels higher than cc). In addition, as a diagnostic method using latex reagents,
In addition to visually determining latex aggregation based on antigen-antibody reactions, we also optically track the decrease in turbidity caused by latex aggregation using visible light, near-infrared light, laser, etc. There is a known method of measuring the antigen or antibody to be measured using a standard curve prepared in advance using antigens or antibodies of known concentrations;
No. 108693 and other publications disclose a measurement method using near-infrared light, but such optical measurement methods also have the same problems as mentioned above, such as aggregation due to non-specific reactions and low sensitivity. Exists. In view of the above-mentioned drawbacks of conventional latex reagents, the present invention was made with the aim of providing a latex reagent that is free from non-specific aggregation and has excellent sensitivity. A latex polymerized in the absence of a persulfate using a polymerization initiator is heated in an alkaline environment, and then the latex is sensitized with an antibody, and then this antibody-sensitized latex is injected into a solution containing serum containing the same antibody as above. The treatment is performed by dispersing the liquid in the liquid and then separating it from the liquid. A method for producing a latex reagent is provided. 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 (SO ₄ ^2- ) at both ends of the molecular chain (polymer chemistry , Vol. 22, No. 244, p. 481 (1965)), and because these sulfate groups are hydrophilic, they are distributed on the surface of the polystyrene particles, and as a result, the polystyrene particles absorb the emulsifier through electrical mutual repulsion. forms a relatively stable latex despite the absence of however,
The sulfate group at the end of the molecular chain is relatively unstable. That is, there is a tendency to form a carboxyl group via a hydroxyl group by hydrolysis. In this case, if the hydrolysis of the sulfate groups remains at the stage of forming hydroxyl groups, the latex becomes unstable because the hydroxyl groups are difficult to dissociate. Therefore, in the present invention, polystyrene having a sulfate group at the end of its molecular chain is heated in alkaline conditions to forcibly hydrolyze substantially all of the sulfate groups into dissociable carboxyl groups, thereby stabilizing the latex. The aim is to The persulfate used in the present invention is not particularly limited, but potassium persulfate, sodium persulfate, ammonium persulfate, etc. are preferably used, and the proportion of these salts used relative to styrene is usually 0.01 to 5% by weight. Polymerization of styrene can be carried out according to the emulsion polymerization method, at 50 to 100°C, preferably at 60°C under a nitrogen stream.
Stir for 5-50 hours at a temperature of ~90°C. 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. to adjust the pH to 7 to 14, preferably 8 to 12, and adjust the pH to 50 to 100°C, preferably 60 to 90.
This is done by heating to a temperature of °C and stirring. 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 for sensitizing the latex particles with antibodies is not particularly limited, and conventionally known methods can be employed as appropriate. For example, latex particles and antibodies are brought into contact with each other at a temperature of about 20 to 37° C. for an appropriate period of time in an appropriate aqueous solvent such as a buffer, physiological saline, or water having a pH of about 7 to 8.7. 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 that are not adsorbed to the latex particles. In the present invention, the sensitized latex prepared above is mixed with a solution containing serum containing the same antibodies as above,
For example, the treatment is carried out by dispersing it in a buffer solution and then separating it from the solution. In this treatment, the sensitized latex is added to a buffer solution containing serum containing antibodies, that is, antiserum at an optimal concentration. Antiserum treatment can be carried out by suspending and stirring for a short time to remove excess antiserum, and then resuspending in a buffer solution, preferably at a concentration of 0.5 to 3% by weight. The concentration of the antiserum in the buffer solution containing the antiserum is preferably 0.1 to 20% by weight, and therefore the concentration of the antibody is preferably about 1 to 1000 μg/cc. Furthermore, the temperature and time during this treatment may be the same as in the case of antibody sensitization. As described above, the latex reagent produced according to the present invention is different from the latex reagent obtained by treating antibody-sensitized latex with an inert protein that does not participate in the antibody-antigen reaction according to the conventional method. Differently, it was treated with serum containing sensitized antibodies, which surprisingly 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 a latex reagent for detection as an example, latex reagents manufactured 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 detects HBs antigen of 10 ng/cc (10 ^-8 g/cc).
It has been found that it is possible to detect antigen amounts up to about 100%, which means that in this case the sensitivity is 1000 times higher. As described above, according to the present invention, it is possible to easily obtain a latex reagent that is free from non-specific aggregation and has extremely high sensitivity. Examples of the present invention will be described below. Example 1 45 g of styrene, 0.044 g of potassium persulfate, and 450 g of ion-exchanged water were charged into a reactor, the polymerization container was purged with nitrogen gas, and polymerization was carried out at a reaction temperature of 70° C. for 30 hours. After the polymerization was completed, the inside of the reaction vessel was replaced with air, the pH of the latex was adjusted to 8.5, and heating was continued at 70°C for 24 hours. The average particle size of the latex thus obtained was 0.38 microns, and the variation in particle size was 0.03 expressed as a coefficient of variation. One volume of this latex was dispersed in a phosphate buffer solution with a pH of 7.4 to give a solid content of 2%, and the solution was passed twice through a column containing an 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 unadsorbed 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, to this precipitated latex, a guinea pig antiserum of 0.1 to 0.1%, which has already absorbed anti-human protein antibodies using a column from the antiserum of a guinea pig immunized with HBs antigen, is added.
The sensitized latex was well dispersed by adding one portion of 5% phosphate buffer, and the mixture was stirred at 37°C for 10 minutes. In phosphate buffer containing this guinea pig antiserum,
The HBs antibody was contained at approximately 1 to 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, Lipaseia (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 Anti-HBs prepared in exactly the same manner as in Example 1, except that the treatment with guinea pig serum containing HBs antibodies in Example 1 was changed to treatment with normal guinea pigs.
When the same test was conducted using antibody-sensitized latex, the results shown in the following table were obtained.

[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. In other words, with this method, non-specific reactions are not so great, but
The detection sensitivity is about 1/1000 of that in the example. Example 2 The latex reagent prepared in Example 1 was sensitized with an alpha-fetoprotein antibody produced by domestic rabbits (a monospecific antibody purified by affinity chromatography), and alpha-fetoprotein antibodies in human serum were sensitized to the latex reagent prepared in Example 1. The agglutination reaction with fetoprotein was investigated. The latex reagent was prepared in the same manner as in Example 1, and was treated with a phosphate buffer containing 1% rabbit antiserum. The results are shown in the table below.

[Table] Additionally, there were only 2 false positives out of 1000 serum samples from normal people. Comparative Example 2 As in Example 2, the latex prepared in Example 1 was sensitized with an 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 the detection sensitivity is only 1/1000 as compared to Example 2. In addition, normal human serum
There were 130 false positives out of 1,000 samples. That is, in the albumin treatment method, the reaction of non-specific aggregation is extremely significant. Example 3 (1) Preparation of anti-HBs antibody sensitized latex (anti-HBs latex) reagent Charge 45 g of styrene, 0.044 g of potassium persulfate, and 450 g of ion-exchanged water into a reactor, replace the polymerization container with nitrogen gas, and adjust the reaction temperature. Polymerization was carried out at 70°C for 30 hours. After the polymerization was completed, the inside of the reaction vessel was replaced with air, the pH of the latex was adjusted to 8.5, and heating was continued at 70°C for 24 hours. The average particle size of the latex thus obtained was 0.38 microns, and the variation in particle size was 0.03 expressed as a coefficient of variation. One volume of this latex was dispersed in a phosphate buffer with a pH of 7.4 to give a solid content of 2%, and the solution was passed twice through a column containing an 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 unadsorbed 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 antibody which has been absorbed in a column was added to the precipitated latex at a concentration of 0.1 to 0.1%.
One volume of 5% phosphate buffer was added to disperse the sensitized latex well, and the mixture was stirred at 37°C for 10 minutes. Anti-HBs in phosphate buffer containing this guinea pig antiserum.
Antibodies were 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 a latex reagent. (2) Optical measurement of latex agglutination reaction 0.5cc of anti-HBs latex reagent prepared in (1) above
was placed in a small test tube, 0.5 cc of phosphate buffer was added thereto, and 1 c.c. of HBs antigen solution with the concentration shown in Table 5 below was added and mixed by shaking for 20 seconds. Acrylic resin cell (light path table 1
cm) and immediately record the change in absorbance over time while stirring at a speed of 200 revolutions per minute. The measurement wavelength is
950nm was used. Next, on this record diagram of the time change of absorbance, draw a straight line along the approximately straight part as soon as possible after the start of recording,
The calculated slope of the straight line is the number in the "Speed" column of Table 5 below, and it shows the change in absorbance.
It is shown in ABS/min.

【table】

[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-fetoprotein (α-FP) antibody sensitized latex (anti-α-FP latex) reagent
-FP antibody (monospecific antibody purified by affinity chromatography) was sensitized. The latex reagent preparation method was the same as in Example 3, and the latex reagent was treated with a phosphate buffer containing 1% rabbit antiserum. (2) Optical measurement of latex agglutination reaction The results shown in Table 6 were obtained in the same manner as in Example 3, using the anti-α-FP latex reagent prepared in (1) above and using 650 nm visible light. .

[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. A latex obtained by polymerizing styrene using persulfate as a polymerization initiator in the absence of an emulsifier is heated in alkaline conditions, and then the latex is sensitized with an antibody. Then, this antibody-sensitized latex is injected with the same antibody as above. 1. A method for producing a latex reagent, comprising dispersing it in a liquid containing serum and then separating it from the liquid.