JPS6116943B2 - - 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 reported to date, 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 discloses 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. Furthermore, 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 have the disadvantage of causing significant non-specific reactions and agglutination with proteins in normal rabbit serum and normal guinea pig serum, and latex self-aggregation due to the properties of albumin. Therefore, the measurement sensitivity is low. 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. . In addition, 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.
At present, it is only possible to detect HBs antigens of approximately (10 ^-5 g/cc) or higher, and there is a need for something with even higher sensitivity. 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. 54−
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 has been made with the aim of providing a latex reagent that is free from non-specific aggregation and has excellent sensitivity. 10% by weight or less of styrene sulfonate is copolymerized in water using persulfate as an initiator in the absence of an emulsifier, and then heated in alkaline conditions to sensitize the resulting latex with antibodies, This antibody-sensitized latex is then treated by dispersing it in a liquid containing serum containing the same antibody as described above and separating it from the liquid. Examples of the styrene sulfonate used in the present invention include sodium styrene sulfonate, potassium styrene sulfonate, lithium styrene sulfonate, and ammonium styrene sulfonate. The ratio of these styrene sulfonates to styrene is 10% by weight or less, preferably 0.0001% to 10%, more preferably 0.0001% to 10%.
It ranges from 0.001% to 5%. Further, examples of the persulfate used as an initiator in the present invention include ammonium persulfate, potassium persulfate, sodium persulfate, and the like. The proportion of these persulfates to the total monomers is preferably in the range of 0.01 to 1% by weight. In the present invention, in order to copolymerize styrene and styrene sulfonate, styrene, styrene sulfonate, and an initiator may be added to a reactor filled with water and heated while stirring. The reaction temperature is usually 50-100℃, preferably 60-85℃
It is preferable to set it in the range of ℃. 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. Further, in the present invention, emulsifiers commonly used in latex production are not used. Next, the latex obtained above is heated under alkaline conditions, and the heating temperature at this time is usually 50 to 100°C, preferably 60 to 85°C, and the heating temperature is 15 to 100 hours. It is better to The above heating is performed by making the reaction system alkaline, and the pH of the reaction system at that time is 7.5 to 12.5.
In particular, it is best to keep it within the range of 8 to 11.5. or,
As mentioned above, the reaction system can be kept alkaline by making it oxygen-free. More specifically, the inside of the polymerization reactor is filled with an inert gas such as nitrogen or helium. This can be achieved by replacing. In this way, a monodisperse latex with an average particle size of 0.05 to 2 microns and a very uniform particle size can be obtained. Although the latex obtained as described above does not contain any emulsifier, it is extremely stable and has a very uniform particle size. The non-specific aggregation reaction caused by the presence of free emulsifier does not occur at all. The reason why the above latex is extremely stable despite not containing any emulsifier can be explained as follows. That is, since persulfate is used as an initiator, sulfuric acid groups (SO ₄ ^-- ) are present at both ends of the polymer chains, and the electrical repulsion caused by these sulfuric acid groups acts between the polymer chains, forming a latex. is stabilized. However, the electrical repulsion caused by the sulfate group at the end of the polymer chain is not sufficient to stabilize the latex. A sufficiently stable latex can only be obtained by adding the electrical repulsion caused by this. What should be mentioned here is:
The aforementioned sulfuric acid group at the end of the polymer chain is relatively unstable and tends to undergo hydrolysis to form a carboxylic acid via the hydroxyl group. In this case, if hydrolysis is incomplete and remains at the stage of forming hydroxyl groups, hydroxyl groups are difficult to ionize and will not contribute to stabilizing the latex. Therefore, the hydrolysis will proceed to a state where most of the hydroxyl groups are carboxyl groups. It is important to stabilize the latex, and it is necessary to carry out the hydrolysis in alkaline conditions to promote the dissociation of carboxyl groups. In the present invention, the reason why the latex is heated under alkaline conditions after the polymerization reaction is to allow the hydrolysis described herein to proceed sufficiently, and this operation produces a highly stable latex. It is possible to obtain. Next, in the present invention, the latex obtained as described above is sensitized with an antibody, but the method of sensitizing the 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 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 containing serum containing antibodies, that is, antiserum at an optimal concentration, suspended for a short time, and stirred.
After removing excess serum, add the buffer preferably
This can be done by resuspending the latex to a concentration of 0.5 to 3% by weight. The concentration of the serum in the buffer solution containing the serum is 0.1 to 20.
% by weight, therefore the concentration of antibody is 1-100μ
It is preferable that it is about g/cc. Further, the temperature and time during this treatment may be the same as in the case of antibody sensitization. As mentioned above, the latex reagent produced according to the present invention differs from the latex reagent produced by treating antibody-sensitized latex with an inert protein that does not participate in antibody-antigen reactions based on the conventional method. Surprisingly, the non-specific agglutination reaction of the latex reagent was effectively removed, and the measurement sensitivity was dramatically increased. As shown in the examples, it has become possible to make accurate diagnoses even for extremely low concentrations of antigens by measuring the agglutination reaction with the naked eye or by using 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
While only HBs antigen of approximately 10 μg (10 ^-5 g/cc) or more can be detected, it has been found that the latex reagent produced based on the present invention can detect antigen amounts of approximately 10 μg (10 ^-8 g/cc). In this case, the sensitivity is actually 1000 times higher. As described above, according to the present invention, it is possible to easily obtain a latex reagent that is highly stable, free from non-specific aggregation, and has extremely high sensitivity. Example 1 91g of styrene, sodium styrene sulfonate
Charge 0.66 g of magnesium sulfate heptahydrate, 0.4 g of potassium persulfate, and 440 g of ion-exchanged water into a reaction container, replace the container with nitrogen gas, and set the reaction temperature to 70°C.
Copolymerization was carried out for 24 hours. After the copolymerization 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 0.51 microns, and the variation in particle size was determined by the coefficient of variation particle size (standard deviation of particle size/
The average particle diameter was 0.04. 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 (antigen immobilized on Sepharose 4B) produced by guinea pigs. (purified product by affinity chromatography) dissolved in phosphate buffer at a concentration of 40 μg/cc was mixed with 1 volume and incubated at 37°C for 2 hours to bind the antibody to the latex. . Next, this sensitized latex was centrifuged at 15,000 rpm for 15 minutes to remove unadsorbed antibodies. The antibody titer in this supernatant is PHA (passive hemagglutination)
It was determined that 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.
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.
In phosphate buffer containing this guinea pig antiserum,
The HBs antibody is 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, 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 the same manner as in Example 1, except that the treatment with guinea pig serum containing HBs antibody in Example 1 was changed to treatment with normal guinea pigs. Got the results.

[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 Example 1. 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 latex reagent was prepared in the same manner as in Example 1, and 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 obtained in Example 1 was sensitized with alpha-fetoprotein antibody, and then washed with a phosphate buffer containing 1% bovine serum albumin to prepare a latex reagent. did. The agglutination reaction with alpha-fetoprotein in human serum was as shown in the table below.

[Table] In this comparative example, 1/1000 of that in Example 2
It is clear that the detection sensitivity is only high. Additionally, 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.

Claims (1)

[Claims] 1 Copolymerizing styrene and styrene sulfonate in an amount of 10% by weight or less based on styrene in water using persulfate as an initiator in the absence of an emulsifier, followed by heating in an alkaline environment. 1. A method for producing a latex reagent, which comprises sensitizing the antibody-sensitized latex, dispersing the antibody-sensitized latex in a liquid containing serum containing the same antibody as described above, and separating the latex from the liquid.