JPH056665B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an improved microcapsule reagent capable of simultaneously detecting multiple antibodies, and a method for producing such a microcapsule reagent. It is known that the reactivity between antigens such as pathogenic bacteria and viruses and antibodies generated thereby is extremely highly specific. In order to discriminate between serotypes by taking advantage of this extremely high serotype specificity, microcapsules sensitized with two or more antigens of different serotypes were used in JP-A-58-21565. According to this method, diagnosis is made based on antigen-antibody reactions that strictly correspond to serotypes, so the accuracy of diagnosis is extremely high. The problem lies in how to equalize the sensitivity levels of different antigen components during sensitization. Having the same sensitivity level is an important condition for improving reproducibility, but since sensitization is performed with a heterologous antigen, it is natural for each sensitivity level to generally vary. This is probably due to differences in the number and activity of bacteria depending on the growth rate and growth conditions of the bacteria themselves. In practice, only those sensitized microcapsules in which each of the antigen components has the same sensitivity level are selected and used, so microcapsules with uneven sensitization were treated as a manufacturing loss. The present inventors have found that even if microcapsules have uneven sensitivity levels, if they are supplemented with microcapsules sensitized with an antigen that lacks sensitivity levels, the uniformity of sensitivity can be improved. We obtained the knowledge that the aggregation pattern became clearer. The mixed microcapsule for detecting multiple antibodies of the present invention comprises a main microcapsule that is sensitized with two or more foreign antigens exhibiting non-uniform sensitivity levels to antibodies, and each antigen has a different agglutination sensitivity to the antibody; It is characterized in that it contains auxiliary microcapsules which are solely sensitized with an antigen that exhibits a low sensitivity level to antibodies. In addition, the method for producing mixed microcapsules for testing multiple antibodies of the present invention involves mixing two or more different antigens that exhibit non-uniform sensitivity levels with respect to antibodies, and then using the sensitized main microcapsules to conduct an antigen-antibody reaction. After this, auxiliary microcapsules are prepared by sensitizing them alone with the antigen for which the sensitivity level was insufficient, and mixed microcapsules are prepared by mixing the main microcapsules and the auxiliary microcapsules. shall be. For example, if the antigen component A has a desired sensitivity level and the sensitivity level of the antigen component B has not reached the former level, after mixing A and B, the antigen component B is The microcapsules sensitized only with the microcapsules are mixed as auxiliary microcapsules. In this mixed microcapsule, an increase in the sensitivity of antigen component B was achieved, but on the other hand, from the perspective of antigen component A, noise increased, and the sensitivity of antigen component A was thought to decrease. However, surprisingly, no desensitization of antigen component A occurred and the initial sensitivity was substantially maintained. Contrary to the above, such an effect is possible even when antigen component A is insufficient at the sensitivity level.
It can be similarly obtained by mixing microcapsules sensitized with component A alone as auxiliary microcapsules. In addition, if only the A component of the main microcapsule has the desired sensitivity level, but the B and C components are insufficient, the B component alone or the C component alone may be sensitized. It may be mixed as auxiliary microcapsules. It is desirable that the auxiliary microcapsules be mixed with the main microcapsules in an amount of 50% by volume or less, preferably in the range of 15 to 45% by volume. If the amount of auxiliary microcapsules exceeds the upper limit, the sensitivity will decrease, and if the amount is too small, the drawbacks of the main microcapsules themselves will become apparent. Sensitizing antigens that can be used in the present invention are selected from a wide range of antigens, including hormones, drug metabolites, specific proteins, viruses, bacteria, cells, and antigens of human origin. Specifically, for example,
These include Treponema pallidum antigen, _HBs antigen, Toxoplasma antigen, Mycoplasma antigen, Shigella, Leptospira, and Vibrio cholerae. Among these, the reagent of the present invention using Leptospira bacteria, which is an antigen component of a different serotype, as a sensitizing antigen is particularly valuable. The amount of these sensitizing antigens is appropriately selected depending on various conditions such as the type thereof and the accuracy of the intended measurement, but generally it is in the range of 0.01 to 10% by weight based on the solid content of the microcapsule. It is within. The method of sensitizing microcapsules with antigens is described in detail in JP-A-58-21565. The microcapsules used as carriers in the present invention consist of a core of an oily substance and a wall material surrounding the core. A typical manufacturing method is, for example, "Microcapsules" by Asashi Kondo, published by Nikkan Kogyo Shimbun (1977).
(2013). For specific oil-based substances, wall materials, various additives, etc., please refer to JP-A-57-
There are detailed descriptions in 196621, 57-19662, etc. Well-known methods are used to sensitize microcapsules with antigens or antibodies, and methods using crosslinking agents are particularly convenient (see "Immobilized Enzymes" by Ichiro Chibata, Kodansha (1975), etc.). Microcapsules used as carriers are 0.85
It has a specific gravity within the range of −1.25, about 0.5 to 20 μm,
Preferably, those having an average particle size in the range from 1 to 10 μm are suitable. Microcapsule carriers usually have a solid content of 1 to
It is desirable to use it within a range of about 3% by weight. The reagent of the present invention is used in immunoassays in which antibodies are detected by observing agglutination images by applying the microtiter method. According to the present invention, it is possible to detect multiple types of antibodies with a single reagent, and furthermore, when observing the immunological cross-reactivity characteristic of the reagent of the present invention, it is possible to detect bacteria with multiple serotype specificities. Infectious diseases can also be accurately diagnosed in just one operation. In the present invention, by using a mixed system of main/auxiliary microcapsules, it is possible to increase production yields, which is very useful industrially. The present invention will be explained in more detail with reference to Examples below. Example 1 Preparation of microcapsule A sensitized with two bacterial strains: 11.8 g of diisopropylnaphthalene and chlorinated paraffin (degree of chlorination 50%, Toyoparax 150)
13.2g of oil (specific gravity approx. 1.10) mixed with 0.25g of oil-soluble red dye Oleosol Red BB (manufactured by Sumitomo Chemical)
was dissolved. The obtained oily substance liquid was added to a solution in which 2.5 g of maleic anhydride-methyl vinyl ether copolymer (GANTREZ AN-149, manufactured by General Aniline and Film Co., Ltd.) was dissolved in 75 ml of water. Stir, emulsify, and coulter counter
The size of oil droplets was measured using a TA-type and adjusted so that the average size was about 5 μm. Add this to 2.5g of urea.
A solution of 0.25 g of resorcinol and 0.3 g of ammonium chloride dissolved in 25 ml of water was added. Plus 50ml of water
Dilute by adding 37% formaldehyde aqueous solution 7
ml was added and reacted at 60°C for 2 hours to perform microencapsulation. Thereafter, a 1N aqueous sodium hydroxide solution was added to adjust the pH to 9.0 to create microcapsules. The microcapsules thus prepared were centrifuged and washed with physiological saline to remove unreacted residues. Microcapsule particle concentration is 10%
The microcapsules were dispersed in physiological saline to give microcapsules A. Preparation of microcapsule reagent A sensitized with two bacterial strains: Take 1.5 ml of the obtained microcapsule A and add 8.5 ml of microcapsule reagent A.
ml of physiological saline was added and dispersed. Next, 100μ of 25% glutaraldehyde aqueous solution
were mixed, reacted for 1 hour at room temperature, and washed by centrifugation.
Redispersed in 10 ml of saline. Leptospira autumnalis strain A strain was grown in Kortov medium (10%
(containing normal rabbit serum) and cultured for 6 to 10
The precipitated bacterial cells obtained by centrifuging the day-old culture solution at 9000 rpm for 20 minutes (5°C) were washed twice with physiological saline. Next, it was suspended in physiological saline and crushed for 10 minutes using a 20kHz sonic crusher (manufactured by Otake Seisakusho). This sonication treatment solution was measured at a wavelength of 280 nm using a spectrophotometer, and the optical density was adjusted to 0.1, which was designated as antigen solution 1. The Leptospira hebdomadeis strain was grown in Kortov's medium in the same manner as the autumn plague A strain. After washing, sonication treatment was performed. The obtained centrifuged supernatant was prepared so that the optical density was 0.1 when measured at a wavelength of 280 nm, and this was used as antigen solution 2. 2 ml each of antigen solutions 1 and 2 were mixed, and this was mixed with 2 ml of the glutaraldehyde-treated microcapsules. After incubating at 37°C for 1 hour, it was left standing in a refrigerator at 4°C for 18 hours. Next, after washing twice with physiological saline containing 0.2% glycine, 2 ml of saline containing 3% bovine serum albumin was added.
Reagent A was obtained by redispersing in 0.15M phosphate buffered saline (PBSPH=7.2). Performance test: The performance of this reagent A was preliminarily tested by the microtiter method using antisera of Autumnalis autumnalis A strain and Hebdomadeis hebdomadeis strain prepared by the method described below. The results obtained are shown in Table 1, in the "Reagent A" column.

[Table] From the above preliminary tests, the desired sensitivity (10240) for anti-Hebdomadeis and Hebdomadeis antibodies was determined.
However, the sensitivity for anti-Autumnalis/Autumnella A antibody was lower than the desired sensitivity (10240).
It was found that this had not been reached. Therefore, in advance, Autumnalis/Autumn Pest A.
was sensitized alone to prepare a single strain sensitization reagent with the desired sensitivity (10240). Mix this with reagent A in a volume ratio of 3:1: Reagent A: Autumnalis Autumn Pest A single sensitization reagent = 3:1.
I got A′. Similar to Reagent A, microtiter tests were performed using the two corresponding antisera. The results obtained are shown in Table 1, in the "Reagent A'" column. Reagent A' of the present invention, which is obtained by mixing reagent A (sensitizing reagent for two strains of Autumnalis and Autumn Plague A and Hebdomades Hebdomadeis strains) with a single sensitization reagent for Autumn nalis and Autumn Plague A, has been tested in a preliminary test. The sensitivity for anti-autumnalis/fallensis A antibody, which had not reached the desired sensitivity, increased to the desired sensitivity. Therefore, the sensitivity level could be made the same for each antibody, and as a result, the detection sensitivity of antibodies was increased without increasing noise. Example 2 Preparation of three bacterial strain sensitized microcapsule reagent B: In the same manner as in Example 1, three bacterial strains, Leptospira autumnalis Autumnitis A strain, Hebdomadeis hebdomadeis strain, Icterohaemoragiae, and RGA strain, were prepared. Grown in Kortov's medium. After washing, each sample was subjected to sonication treatment. 280nm
Each sonication treatment solution prepared so that the optical density showed 0.1 at the wavelength of was used as the antigen solution. Reagent B was obtained by mixing 2 ml of antigen solutions of three types of bacterial strains and reacting them in microcapsules in the same manner as in Example 1. As in Example 1, a preliminary test was conducted using antisera corresponding to three types of bacterial strains by the microtiter method. The results obtained are shown in Table 2, column "Reagent B".

[Table] Preliminary test results, anti-autumnalis, autumn plague A
Although we were able to obtain the desired sensitivity (10240) for the antibody, the sensitivity for anti-Icterohaemoragiae RGA antibody and anti-Hebdomadeis antibody did not reach the desired sensitivity (10240). I understand. Therefore, in advance, Icterohemoragie
A single strain sensitization reagent having the desired sensitivity (10240) was prepared by sensitizing RGA and Hebdomadeis individually. This product was mixed with reagent B in a volume ratio of reagent B: Icterohemoragie RGA single reagent: Hebdomadeis hebdomadeis strain single reagent = 3:1:1 to obtain reagent B'. Similar to reagent B, a microtiter test was performed using three antisera. The results obtained are shown in Table 2, in the "Reagent B'" column. The sensitivities for anti-Icterohaemoragiae RGA antibody and Hebdomadeis hebdomadeis antibody, which had not reached the target sensitivity (10240) in the preliminary test, were increased to the target sensitivity with reagent B' of the present invention. Therefore, the sensitivity level for each antibody was the same, and as a result, three types of antibodies could be detected simultaneously with good reproducibility and high accuracy without increasing noise. Microplate test using antiserum: Reagents A, A', B, prepared in Examples 1 and 2
The performance of B' was evaluated by antibody titer using the microtiter method using the following antiserum. (Preparation of antiserum) (a) Leptospira icterohemoragiae RGA
rabbits were hyperimmunized with each of the strains (a), (b), and (c) above to prepare antisera. The Kortov medium culture solution of each bacterial strain was centrifuged, and the precipitated bacterial bodies were suspended in physiological saline.
This was subcutaneously injected into rabbits twice at an interval of 4 to 5 days.
Nine additional intravenous injections were given at 4-5 day intervals. Seven to eight weeks have passed since the first subcutaneous injection, and after confirming that the prescribed antibody titer has been obtained, whole blood is collected.
Antiserum for each strain was prepared. (Test by microtiter method) Reagents A, A', sensitized to Leptospira bacterial cell components
For B and B', antigen-antibody reactions were carried out using the microtiter method. Tubes in which clear agglutination was observed were considered positive, and the highest dilution factor of the antiserum against the three types of bacterial strains was determined, which was used as the antibody titer. For the antisera of the three types of bacterial cells, dilute 25μ of the test serum into each tube hole of the microplate at 2-fold intervals using 0.15M phosphate buffered saline (PBSPH7.2), and perform a multiple dilution series. It was created. Next, 25 µm each of the microcapsule reagents A, A', B, and B' sensitized with the Leptospira cell components were collected with a dropper and sequentially dropped into the tube holes of the antiserum dilution series of the microplate. After shaking the microplate for 5 minutes to advance the antigen-antibody reaction,
It was left in the refrigerator at ℃ for 18 hours. Then take it out,
The microplate was placed on a light table and the agglutination image at the bottom of the tube was observed, and the highest dilution of the serum that showed agglutination was taken as the antibody titer.

Claims (1)

[Scope of Claims] 1. Main microcapsules sensitized with two or more foreign antigens exhibiting non-uniform sensitivity levels to antibodies, and each antigen having a different agglutination sensitivity to antibodies, and one of the foreign antigens having a lower sensitivity level to the antibodies. A mixed microcapsule for detecting multiple antibodies, characterized in that it contains an auxiliary microcapsule that is singly sensitized with an antigen that exhibits a sensitivity level. 2. The mixed microcapsule according to claim 1, wherein the foreign antigen is an antigen component of a different serotype. 3. The mixed microcapsule according to claim 2, wherein the antigen component is Leptospira bacteria. 4 After performing an antigen-antibody reaction using the main microcapsules that have been sensitized after mixing two or more foreign antigens that exhibit non-uniform sensitivity levels to antibodies, a single sensitization test is performed using the antigen for which the sensitivity level was insufficient. A method for producing mixed microcapsules for testing multiple antibodies, comprising: preparing auxiliary microcapsules by preparing auxiliary microcapsules, and mixing the main microcapsules and the auxiliary microcapsules to prepare mixed microcapsules. 5. The manufacturing method according to claim 4, wherein the foreign antigen is an antigen component of a different serotype. 6. The manufacturing method according to claim 5, wherein the antigen component is Leptospira bacteria.