JP2735642B2 - Antibody-immobilized insoluble carrier particles - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an immunological measurement reagent, and more particularly, to an insoluble carrier particle on which an antibody is immobilized.

[Problems to be Solved by the Prior Art and the Invention] Conventionally, immunologically active substance-immobilized insoluble carrier particles (hereinafter, immobilized carrier particles) in which an antibody is immobilized on the insoluble carrier particles by physical adsorption or formation of a covalent bond. Abbreviated) and the corresponding antigen in the subject, such as serum or urine, by observing the agglutination reaction or the agglutination inhibition reaction based on the antigen-antibody reaction to measure the corresponding antigen in the subject. Measurement methods are known. In order to use this immunological measurement method for clinical diagnosis, it is necessary to use an antibody that does not recognize a variety of antigens in a subject other than the antigen to be measured. In clinical diagnosis, it has been conventionally required to specifically measure only a single antigen. Therefore, only one antibody that recognizes only a single antigen was immobilized on insoluble carrier particles and used. The measurement method using the above-described immobilized carrier particles is widely used because a trace amount of antigen contained in a specimen can be measured quickly, accurately, and easily.

However, in the case where the antigen is, for example, a bacterium, a virus, or a fungus, it has conventionally been mainly detected by a culture method.
Measurement by this culture method required several days to make a determination, and it was difficult to make a quick diagnosis.

When the above-mentioned immobilized carrier particles are used, the determination can be made in a few minutes, so that a diagnosis can be made on the spot. For this reason, attempts have been made to produce various immobilized carrier particles and use them for diagnosis.

For example, for the purpose of diagnosing vaginal candidiasis, anti-Candida albicans corresponding to the causative fungus Candida albicans
Anti-Candida albicans with antibody immobilized on insoluble carrier particles
When antibody carrier particles are prepared and used, Candida al
High sensitivity to bicans.

However, it is another causative agent of vaginal candidiasis and anti-Cand
The sensitivity is low for Candida glabrata where the ida albicans antibody shows a cross-reaction, and it does not reach practical sensitivity. Conversely, when anti-Candida glabrata antibody-immobilized carrier particles are prepared and used, the sensitivity to Candida albicans is similarly low, and does not reach practical sensitivity. Therefore, anti-Candida al
Even with the use of immobilized carrier particles on which either the bicans antibody or the anti-Candida glabrata antibody is immobilized, accurate diagnosis of vaginal candidiasis cannot be made. In addition, anti-Candida albicans antibody-immobilized
When the carrier particles are simply mixed with Candida glabrata antibody-immobilized carrier particles, the sensitivity becomes insufficient for any antigen.

Furthermore, the sensitivity of antigen recognition can be increased by increasing the degree of purification of the antibody used. However, if this method is used, the sensitivity to the causative bacterium of vaginal candidiasis, that is, the above-mentioned two antigens only increases. However, it has a drawback in that it shows cross-reactivity to bacteria other than the causative bacteria of vaginal candidiasis, and the specificity for vaginal candidiasis decreases. That is, the sensitivity and specificity of the conventional detection method of vaginal candidiasis using antibody-immobilized insoluble carrier particles are insufficient compared with conventional detection methods such as culture methods, and the sensitivity and specificity can be satisfied simultaneously. Did not. There is a need for antibody-immobilized carrier particles that simultaneously satisfy sensitivity and specificity and enable accurate diagnosis in the diagnosis of multiple diseases caused not only by vaginal candidiasis but also by bacteria, viruses, and fungi. Was.

By the way, when the antigen-immobilized carrier particles prepared by immobilizing the monoclonal antibody prepared for a single antigen on the carrier particles and the antigen do not show agglutination, when the antigen is not aggregated, the same antigen can be used at different antigen determination sites. It is known that when two or more monoclonal antibodies prepared in combination are immobilized in combination, the antibody-immobilized carrier particles may mix with the antigen to cause aggregation. That is, the conventional technique is to immobilize two or more antibodies against a single antigen.

On the other hand, the present invention has a different technical idea from the above-mentioned conventional technology,
This study was conducted for the purpose of recognizing and aggregating two or more different antigens with good sensitivity and specificity.

[Means for Solving the Problem] The present inventors have intensively studied to obtain antibody-immobilized carrier particles that can achieve the above object, and as a result, immobilized at least two kinds of antibodies that recognize different antigens. When the immobilized carrier particles were used clinically for diagnosis, they found that both sensitivity and specificity were excellent, and completed the present invention.

That is, the present invention provides an immunological method comprising immobilizing at least two types of antibodies that recognize different antigens and also have cross-reactivity with antigens recognized by other antibodies on the same insoluble carrier particles. It is an antibody-immobilized insoluble carrier particle for agglutination measurement.

In the present invention, a plurality of antibodies are immobilized on insoluble carrier particles. The antibody to be immobilized, at least two of the plurality of antibodies recognize different antigens from each other,
There is no particular limitation on the combination of antibodies having cross-reactivity with each other, and two or more known antibodies can be used in combination.

For combinations of different antigens, for example, a combination when there is clinical significance to simultaneously measure bacteria or viruses of the same genus, or a clinical measurement purpose even if different antigens do not have close taxonomic relationships. Combinations in which there is a common feature and the significance of simultaneous measurement are particularly preferable combinations of the present invention in the present invention.

Representative examples that can be suitably used include, for example, anti-Candida albicans antibody / anti-Candida glabrata antibody;
Candida albicans antibody / anti-Candida glabrata antibody / anti-Tr
achomonas antibody; anti-Mycoplasma pneumoniae antibody / anti-Myco
and a combination of two or more antibodies corresponding to antigens classified into the same genus in bacteria, viruses, fungi, and the like.

The combination of the immobilized antibodies is a combination of antibodies in which at least two kinds of antibodies cross-react with each other in an antigen corresponding to another antibody.

Therefore, the present invention also provides the antibody-immobilized insoluble carrier particles for immunoagglutination measurement according to claim 1, wherein at least two kinds of antibodies recognizing different antigens have cross-reactivity with antigens recognized by other antibodies. provide.

1976, Medical Immunology, published by Nankodo Co., Ltd., No. 117
Pages -118 describe cross-reactivity as follows. Anti-A generated by immunization with A or B antigen
Alternatively, B antibody reacts only with each A or B antigen, and A antigen does not react with anti-B, C, D... Antibodies. (Omitted) This principle can sometimes be broken. For example, anti-bovine serum albumin antibodies can also react to some extent with sheep serum albumin. Such a reaction is called a cross reaction. In principle, the cross-reaction is weaker than this reaction, and the more closely related the species from which the antigen is derived, the stronger it appears. This is a phenomenon that occurs because both albumins have a common determinant.

In the present invention, the cross-reactivity, which will be described later in detail, shows that the immobilized carrier particles on which a single antibody is immobilized react with an antigen other than the antigen corresponding to the immobilized antibody and agglomerate. That is.

Conventionally, a technique is known in which two types of monoclonal antibodies prepared for different antigen determination sites of a single antigen are immobilized on carrier particles. However, in this case, the only antigen showing aggregation is the single antigen. On the other hand, the present invention is characterized in that at least two kinds of antibodies are antibodies showing agglutinating reactivity to different antigens, respectively. It differs from the embodiment of the present invention in that it is only an antigen.

The insoluble carrier particles are insoluble carrier particles that are substantially insoluble in the liquid medium used for immobilization, storage and measurement. Fine particles having an average particle diameter of about 10 μm or less, which will be described in detail later, are preferably used.

Various types of these particles are already known for use in antigen-antibody reactions, and in the present invention, these known fine particles can be used without any particular limitation. Particularly preferred examples include those obtained by an emulsion polymerization method such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer. Fine particles of an organic polymer substance such as an organic polymer latex particle, or an inorganic oxide such as silica, silica-alumina, or alumina, or an inorganic particle obtained by introducing a functional group into the inorganic oxide by an operation such as silane coupling treatment. Further, there are biological particles such as human O-type red blood cells and sheep red blood cells.

Regarding the particle size of the insoluble carrier particles, when the particle size is large, the amount of change in the particle size due to aggregation is large but the agglutination reaction speed is slow, and when the particle size is small, the Brownian motion is active and the agglutination reaction speed is fast but the primary Since the particle diameter is small, the amount of change in the particle diameter due to the aggregation reaction is small. For this purpose, the average particle size of the insoluble carrier particles used in the agglutination reaction is 10
Insoluble carrier particles of about μm or less, preferably 0.05 to 5.0 μm, are suitably used.

In the present invention, the immunological measurement method using the antibody-immobilized carrier particles, that is, an agglutination reaction or aggregation inhibition based on an antigen-antibody reaction between the antibody on the antibody-immobilized carrier particles and the corresponding antigen or the like in the subject To observe the reaction,
Known methods such as visual and optical measurement methods are not particularly limited and can be used.

In the present invention, the method of immobilizing the antibody on the insoluble carrier particles may be either physical adsorption or formation of a chemical covalent bond. Many methods for forming a chemical covalent bond have already been proposed, and an immobilization method may be selected from known methods according to the characteristics of the antibody to be immobilized. Generally, the antibody may be mixed with the insoluble carrier particles in a dispersion medium in the presence of a crosslinking agent, if necessary. Known crosslinking agents such as glutaraldehyde and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can be used.

The dispersion medium for immobilizing the antibody on the insoluble carrier particles is not particularly limited, and a known dispersion medium may be used. However, when the above-described crosslinking agent is used, the dispersion medium in which the components in the dispersion medium do not react with the crosslinking agent is used. It is necessary to use a medium. As a suitable dispersion medium, a buffer such as a phosphate buffer, a glycine-sodium hydroxide buffer, a Tris-hydrochloride buffer, or an ammonium chloride-ammonia buffer is preferably used. The concentration of the insoluble carrier particles in the dispersion medium during immobilization is not particularly limited, but generally, at the time of mixing with the antibody.
It is suitable to select 0.05% by weight or more, preferably 0.2 to 2.0% by weight. The concentration of the antibody is not particularly limited, but generally, at the time of mixing with the insoluble carrier particles.
It is suitable to select 0.0005% by weight or more, preferably 0.002 to 0.2% by weight.

In the present invention, it is essential that at least two kinds of antibodies are combined and immobilized.

When combining two types of antibodies, it is necessary that both antibodies have cross-reactivity with each other to an antigen corresponding to the other antibody. When combining three or more antibodies,
Any one of the two antibodies may have cross-reactivity with an antigen corresponding to the other antibody. Of course, each antibody may have cross-reactivity with all antigens corresponding to other antibodies.

As a method for confirming that an antibody has cross-reactivity with an antigen corresponding to another antibody, for example, a type of antibody (antibody A) is immobilized on insoluble carrier particles to produce antibody A-immobilized carrier particles, and immobilization is performed. An agglutination reaction is carried out with the antigen A corresponding to the antibody A which has been converted, and the minimum concentration of the antigen in which agglutination is recognized, that is, the sensitivity is determined. Next, the same antibody A-immobilized carrier particles are mixed with an antigen B corresponding to another antibody B, and the presence or absence of cross-reactivity is confirmed based on the presence or absence of agglutination. The sensitivity of the antibody A-immobilized carrier particles to the antigen B is determined in the same manner. As the antigen corresponding to each antibody, the antigen used when producing the antibody may be used. Conversely, the sensitivity of the antibody B-immobilized carrier particles to antigens B and A is determined in the same manner.
In the present invention, a combination in which each antibody shows cross-reactivity with an antigen corresponding to another antibody can be particularly preferably used.

The sensitivity ratio of the antibody-immobilized carrier particles determined by the agglutination reaction to each antigen, that is, the ratio of the lowest concentration showing aggregation is not particularly limited, but the sensitivity to the antigen corresponding to the antibody is set to 1 and the sensitivity to other antibodies is determined. The sensitivity to the antigen is particularly preferably 100 or less.

The order in which two or more antibodies are combined and immobilized is not particularly limited, and even if two or more antibodies are simultaneously immobilized,
It may be fixed sequentially. The ratio of the amounts used of the plurality of antibodies is not particularly limited, and may be determined in consideration of the sensitivity, specificity, etc. of the obtained immobilized carrier particles. It is preferable to use 30% or more.

[Operation and Effect] A comparison between the method for producing the antibody-immobilized carrier particles according to the present invention and the conventional method for producing the antibody-immobilized carrier particles for, for example, the diagnosis of vaginal candidiasis shows that the antibody-immobilized carrier particles according to the present invention are as follows. Is excellent.

An accurate diagnosis cannot be made by a conventional detection method using antibody-immobilized carrier particles on which the primary antibody is immobilized. That is, an anti-Candida albica immobilized with an anti-Candida albicans antibody corresponding to Candida albicans, the causative bacterium of vaginal candidiasis
The ns antibody-immobilized carrier particles cannot be detected with practical sensitivity to Candida glabrata, another causative agent of vaginal candidiasis. Conversely, when carrier particles on which anti-Candida glabrata antibody are immobilized are prepared, Candida albicans cannot be detected with practical sensitivity. In comparison, the antibody-immobilized carrier particles according to the present invention, that is, the anti-Candida albicans antibody and the anti-Candida albicans antibody
According to the immobilized carrier particles on which both the Candida glabrata antibody are immobilized, immobilized carrier particles exhibiting practically high sensitivity to both Candida albicans and Candida glabrata can be obtained.

In addition, anti-Candida albica prepared separately in advance
When the ns antibody-immobilized carrier particles and the anti-Candida glabrata antibody-immobilized carrier particles are simply mixed, the sensitivity becomes insufficient for any antigen, which is not practical. Regarding this cause, the present inventors consider as follows. That is, when two types of immobilized carrier particles on which a single antibody is immobilized are mixed, each immobilized carrier particle strongly reacts only with the corresponding antigen and weakly reacts with the other antigen, and Since the immobilized carrier particles having a weak reaction coexist, the frequency of collision of the immobilized carrier particles that should originally strongly aggregate is reduced, and as a result, the sensitivity becomes insufficient for any antigen. On the other hand, the antibody-immobilized carrier particles according to the present invention have two
Species antibodies coexist and react strongly to any antigen. Therefore, unlike the case where two kinds of the above-described immobilized carrier particles are mixed, there is no phenomenon in which the frequency of collision between the immobilized carrier particles is reduced, and it is considered that the antibody exhibits high sensitivity to any antigen.

In conventional antibody-immobilized carrier particles using only a single antibody, increasing the degree of purification of the antibody used can increase the sensitivity to each other's antigens. For example, even in the case of vaginal candidiasis, cross-reactivity to bacteria other than the causative bacterium is exhibited, and there is a disadvantage that specificity is reduced. In contrast, in the antibody-immobilized carrier particles of the present invention, since two types of antibodies coexist on the same immobilized carrier particles, unnecessary cross-reactivity is diluted by the other antibody, and the decrease in specificity is suppressed. Think of it. That is, the conventional antibody-immobilized carrier particles have insufficient sensitivity and specificity as compared with conventional detection methods such as a culture method, and cannot simultaneously satisfy the sensitivity and specificity required for use in diagnosis. . In contrast, the antibody-immobilized carrier particles of the present invention satisfy sensitivity and specificity at the same time and can be used as various diagnostic reagents that require accuracy.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Examples 1-4 Candida reagent (1) Preparation of immunizing antigen Candida albicans c, which is one of the causative bacteria of candidiasis
-S-111 (obtained from Chiba Univ.
Inoculate yeast extract peptone dextrose medium, 25 ℃
For 24 hours. 1/10 volume of formalin (37% formalin solution) was added to the cell fluid after culturing, the mixture was treated at room temperature for 30 minutes to kill cells, and then centrifuged (1000 × g, 10 minutes).
Then, 1/60 mol of phosphate buffered saline (hereinafter abbreviated as PBS, pH 7.2) was added to the sediment (cells) and washed. The operations of centrifugation and washing were further repeated twice. The resulting precipitate was added physiological saline to give the immunizing antigen to prepare a concentration of bacteria of 10 ⁸ cells / ml.

Candida glabra, another causative agent of candidiasis
tat-g-108 (also obtained from the Chiba Univ.Eukaryotic Microorganisms Center) was operated in the same manner as the above Candida albicans cs-111 to obtain an immunizing antigen whose bacterial concentration was adjusted to 10 ⁸ cells / ml. .

(2) Production of antibody Candida albicans c-a-1 obtained in (1) was added to New Zealand white rabbit (female, weight about 2 kg).
Eleven immunization antigens were intravenously injected 1 ml at a time every other week for a total of 4 times. Seven days after the final immunization, partial blood was collected, booster immunization was further performed, and after 7 days, whole blood was collected. Anti-Candida albicans antiserum was obtained from blood obtained by partial blood sampling and whole blood sampling.

The same procedure was used for the immunizing antigen of Candida glabrata t-g-108 to obtain an anti-Candida glabrata antiserum.

(3) Preparation of affinity purified antibody (3-a) Preparation of purified mannan antigen The immunizing antigen of Candida albicans c-a-111 obtained in (1) was centrifuged and then freeze-dried. Add water to this 10
% (W / V), autoclaved (121 ° C, 3
After aging), acetic acid was added to 1 N. This solution was centrifuged to obtain a supernatant containing the mannan antigen. An aqueous solution of sodium hydroxide was added to the supernatant for neutralization. One volume of the neutralized solution was added with 3 volumes of ethanol to precipitate mannan antigen, and centrifuged to obtain a sediment containing the mannan antigen. The precipitate was washed with an 80% aqueous ethanol solution, and then centrifuged again to obtain a precipitate containing a mannan antigen.
Add 30 times the weight of water to the weight of the wet sediment and dissolve it.
After stirring overnight, the mixture was centrifuged to obtain a sediment containing the mannan antigen. Water was added 10 times the weight of the sediment to suspend it, and concentrated hydrochloric acid was added 5 times the same weight to dissolve the sediment. Thereafter, the operation of precipitating the mannan antigen with ethanol and dissolving with water was repeated twice, and an ion exchange resin (Amberlite CG-120) was added, followed by stirring at 4 ° C. for 2 hours. Then, filtration was performed, and 0.1 M sodium chloride and then ethanol were added to the filtrate to precipitate purified mannan antigen. A precipitate was obtained by centrifugation, and water was added thereto to obtain a purified mannan antigen aqueous solution.

Candida al obtained by gas chromatography
It was confirmed that the purified mannan antigen of bicans consisted only of mannose. The sugar concentration determined by the anthrone-sulfuric acid method was 60 mg / ml.

The same procedure was applied to the immunizing antigen of Candida glabrata t-g-108 obtained in (1) to obtain a purified mannan antigen of Candida glabrata. The sugar concentration was 26 mg / ml.

(3-b) Preparation of Sepharose Coupling Purified Mannan Antigen Epoxy-activated Sepharose 6B (Pharmacia LKB Biotechnology) and Candida obtained in (3-a)
albicans purified mannan antigen under alkaline conditions (pH 1
After mixing in 2) and coupling the purified mannan antigen, ethanolamine was added to inactivate unreacted epoxy groups, and the mixture was equilibrated with PBS to obtain Sepharose to which the purified mannan antigen of Candida albicans was coupled.
The same procedure was applied to the purified mannan antigen of Candida glabrata obtained in (3-a) to obtain sepharose to which the purified mannan antigen of Candida glabrata was coupled.

(3-c) Affinity purification of antibody using purified mannan antigen-coupled sepharose An equal volume of saturated ammonium sulfate was added to the anti-Candida albicans antiserum obtained in (2), and the mixture was stirred at 4 ° C for 10 hours, and the resulting precipitate was centrifuged. separated. Next, a solution obtained by adding an equal volume of water to the antiserum to the precipitate and dissolving it was dialyzed against water, and further dialyzed against PBS. After dialysis, Candida albi obtained in (3-b)
Sepharose coupled with purified mannan antigen of cans was added, and the mixture was stirred at 4 ° C. for 16 hours. Thereafter, a column was prepared, and unreacted antibodies (antibodies having no antibody activity to mannan) were distilled off. After sufficient washing with PBS, 0.05M glycine hydrochloride buffer (pH 2.2) was added to elute the reacted antibodies (antibodies having antibody activity against mannan), immediately neutralized with Tris buffer, and then subjected to anti-Candida albicans An affinity purified antibody was obtained. The Candida glabrata antiserum obtained in (2) was treated in the same manner using Sepharose to which purified mannan antigen of Candida glabrata was coupled, and the anti-Ca
A ndida glabrata affinity purified antibody was obtained.

(4) Preparation of antibody-immobilized carrier particles Anti-Candida albicans and anti-Candida glab obtained in (3)
Rata affinity purified antibodies were separately dialyzed against 1 / 60M phosphate buffer (hereinafter abbreviated as PB, pH 6.3),
Was adjusted to a concentration of 0.25 mg / ml, and as Example 1, each was mixed at a volume ratio of 3 to 2 to obtain an antibody mixture. Further, as Examples 2 to 4, each was mixed at a volume ratio of 4: 1, 2: 3, 1: 4 to obtain antibody mixtures. Next, polystyrene latex particles (carrier particles) having an average particle size of 0.728 μm were diluted with PB to prepare a dispersion having a latex concentration of 1% by weight, and an equal volume was added to the above antibody mixture, followed by stirring and mixing immediately. After standing for 2 hours, 0.1 part by weight of bovine serum albumin (lyophilized product) was added,
Let stand for hours. Subsequently, the mixture was centrifuged and the resulting precipitate (antibody-immobilized carrier particles) was suspended by adding twice the volume of PB to the antibody solution (particle concentration: about 0.5% by weight).
A dispersion of antibody-immobilized carrier particles, in which both the s affinity purified antibody and the anti-Candida glabrata affinity purified antibody were immobilized, that is, a Candida reagent was obtained.

(5) Agglutination reaction (5-1) Sensitivity test Candida albicans ca-111 and Candi obtained in (1)
Each immunizing antigen prepared at a bacterial concentration of 10 ⁸ cells / ml of da glabrata t-g-108 was diluted with PB to a bacterial concentration of 100 × 10
⁵ , 50x10 ⁵ , 25x10 ⁵ , 12.5x10 ⁵ , 6.3x10 ⁵ , 3.
1x10 ⁵ cells, 1.6 × 10 ⁵ cells were prepared 0.8 × 10 ⁵ pieces of antigen solution.
Each of the Candida reagents of Examples 1 to 4 obtained in (4) was dropped on a glass plate one by one, and the above Ca was added to each one drop of the Candida reagent.
ndida albicans and Candida glabrata antigen dilution 1
The drops were added, mixed with a toothpick, and immediately stirred horizontally by rotating the glass plate horizontally at a speed of 120 rotations per minute for 2 minutes using a Teher type stirrer manufactured by Hirasawa Seisakusho Co., Ltd. The state of aggregation after stirring was visually determined, and the sensitivity of each Candida reagent to each antigen was determined using the minimum concentration of the antigen in which aggregation was clearly observed. The results obtained are shown in Table 1.

(5-2) Specificity test Candida albicans cs-111 and Candida glabrata
Instead of tg-108, other fungi of the same genus Candida and fungi of other genus (obtained from the Center for Eukaryotic Microorganisms, Chiba), and the resident bacteria present in the specimen when using the Candida reagent ( 1m after dilution with PB)
The bacterial cell fluid was prepared so that the bacterial concentration per l was 100 × 10 ⁵ cells.

Each of the Candida reagents of Examples 1 to 4 obtained in (4) was dropped on a glass plate one drop at a time, and one drop of each of the bacterial cell liquids was added to one drop of each Candida reagent. (5-1) Sensitivity test A cohesion test was performed in the same manner as in the above. The state of aggregation is determined with the naked eye. When no aggregation is observed (−), when it is difficult to determine the presence or absence of aggregation (±), or when aggregation is clearly observed, (++), (++) ) And (+). The results obtained are shown in Table 1.

Comparative Examples 1-2 In Example 1, anti-Candida albicans and anti-Candid
a glabrata affinity-purified antibody was mixed with each antibody solution, and instead of using as an antibody mixture, anti-Candida albi
A dispersion of antibody-immobilized carrier particles in which an anti-Candida albicans affinity purified antibody was immobilized alone was obtained in the same manner as in Example 1 except that the cans affinity purified antibody solution was used alone. Comparative Example 1 An anti-Candida glabrata affinity purified antibody solution was used in the same manner as in Example 1 except that the antibody solution was used alone.
A dispersion of antibody-immobilized carrier particles in which Candida glabrata affinity-purified antibodies were immobilized alone was obtained. Comparative Example 2 An agglutination reaction was performed in the same manner as in Example 1. The results obtained are shown in Table 1.

As shown in the sensitivity test results in Table 1, Comparative Examples 1 and 2
Antibodies Antibodies immobilized immobilized singly carrier particles of, and cell concentration 1.6 respectively for each of the immunizing antigen 6.3X10 ⁵ cells / m
l of antigen diluent It showed aggregation and was highly sensitive. In contrast, for antigens corresponding to other antibodies, bacterial concentrations of 50 and 12.5x1 respectively
0 ⁵ / ml antigen dilution simply represents aggregation was less sensitive.

On the other hand, the antibody-immobilized carrier particles in which the two antibodies of Examples 1 to 4 were both immobilized were prepared with an antigen diluent having a bacterial concentration of 1.6 and 6.3 × 10 ⁵ cells / ml for each of the immunizing antigens. It showed aggregation and was highly sensitive. That is, the antibody-immobilized carrier particles of the present invention show the same high sensitivity as the antibody-immobilized carrier particles (Comparative Examples 1 and 2) on which the antibody is immobilized alone show the respective immunity antigens. Examples 2 and 3 show the sensitivity to antigens corresponding to other antibodies in Examples 1 to 2.
All four were eight times more sensitive to each antigen.

The results of the specificity test in Table 1 show that Candida albicans ca-104 and Candi as antigens of the same species as the respective immunizing antigens.
Aggregation reactivity with da glabrata t-g-109 was shown.
As shown in Table 1, the antibody-immobilized carrier particles on which the antibodies of Comparative Examples 1 and 2 were individually immobilized exhibited strong agglutination indicated by (++) with respect to the same species of antigen as each immunizing antigen. On the other hand, for the antigens of the same species as the antigens corresponding to the other antibodies, it was difficult to determine the presence or absence of weak aggregation indicated by (+) and aggregation indicated by (±), respectively.
On the other hand, the antibody-immobilized carrier particles of the present invention clearly show aggregation (+++,
++).

Next, antigens that differ in species from the respective immunizing antigens but have the same genus, bacteria that are different in genus but are the same fungi, and bacteria that are different in genus, which are usually present in a sample to be measured for a Candida reagent, are so-called normal antigens. The agglutination reactivity with bacteria was observed. Comparative Example 2
Shows non-specific aggregation and poor specificity, whereas the antibody-immobilized carrier particles of the present invention did not show any aggregation (±,
-) Excellent specificity.

(5-3) Measurement of patient sample (5-3-1) Measurement by culture method From a patient suspected of candidiasis, vaginal secretion was collected with a cotton swab, and chloramphenicol-added Sabouraud glucose agar medium (Difco) After incubation at 37 ° C for 48 hours.
A specimen in which more than one colony was recognized was determined to be positive for candidiasis.

(5-3-2) Measurement using antibody-immobilized carrier particles In (5-3-1), a secretion fluid collected with a cotton swab from a patient determined to be positive for candidiasis by the culture method is dispersed in 1 ml of PB and used as a sample. did.

(5-1) An aggregation test was performed in the same manner as the sensitivity test. The ratio (positive rate) of the sample in which aggregation was clearly observed was determined for Examples 1 to 4 and Comparative Examples 1 to 2 for the samples of 10 patients determined to be positive for candidiasis by the culture method. .

While the positive rates of Examples 1 to 4 all showed practically sufficient sensitivity of 100%, the positive rate of Comparative Example 1 was 80%,
In Comparative Example 2, the positive rate was inferior to 60%, which was not suitable for practical use.

As described above, the Candida reagent using the antibody-immobilized carrier particles of the present invention is Candida, a causative bacterium of candidiasis.
While high sensitivity was shown for both albicans and Candida glabrata, Comparative Examples 1 and 2 were low in sensitivity to antigens corresponding to other antibodies. As for the patient sample, the Candida reagent using the antibody-immobilized carrier particles of the present invention showed a high positive rate and showed sufficient sensitivity for practical use, whereas Comparative Examples 1 and 2 had a low positive rate and were not suitable for practical use. Was. Furthermore, regarding the specificity, the reagent of Comparative Example 2 showed clear aggregation even for antigens other than the antigen corresponding to each antibody, and the specificity was poor.

This means that when the reagent of Comparative Example 2 was clinically used, nonspecific agglutination was frequently observed even for specimens other than candidiasis, meaning that it was not practical.

On the other hand, the Candida reagent using the antibody-immobilized carrier particles of the present invention was excellent in specificity.

Examples 5 to 7 (1) Preparation of DEAE-purified antibody An equal volume of saturated ammonium sulfate was added to the high Candida albicans antiserum obtained in (1) of Example 1, and the mixture was stirred at 4 ° C for 1 hour. Centrifuged. Next, a solution obtained by adding an equal volume of water to the antiserum to the precipitate and dissolving it was dialyzed against water, and further dialyzed against PB. The dialysate is passed through a DEAE Sepharose column equilibrated with PB, and the flow-through fraction is collected and the anti-Ca
A DEAE purified antibody of the ndida albicans antibody was obtained.

The same procedure was used for the anti-Candida glabrata antiserum to obtain a DEAE-purified anti-Candida glabrata antibody.

(2) Preparation of antibody-immobilized carrier particles Anti-Candida albicans DEAE purified antibody obtained in (1)
PB was separately added to each of the purified antibodies of Candida glabrata DEAE, and each was adjusted to a concentration of 0.50 mg / ml. As Example 5, each was mixed at a volume ratio of 1: 1 to obtain an antibody mixture.

Further, as Examples 6 and 7, antibody mixtures were obtained by mixing at a volume ratio of 9: 1 and 1: 9, respectively. Using the same carrier particles as those used in Example 1, the same operation as in Example 1 was carried out, and a dispersion of the antibody-immobilized carrier particles in which the anti-Candida albicans DEAE purified antibody and the anti-Candida glabrata DEAE purified antibody were both immobilized. That is, a Candida reagent was obtained.

(3) Aggregation reaction An agglutination reaction was performed in the same manner as in Example 1 for Examples 5 to 7. The results obtained are shown in Table 2.

Comparative Examples 3 to 5 In Example 5, anti-Candida albicans and anti-Candid
a Glabrata DEAE-purified antibody was mixed, and the same procedure as in Example 5 was repeated except that the anti-Candida albicans DEAE-purified antibody solution was used alone instead of using the antibody mixture as an antibody mixture. A dispersion of the antibody-immobilized carrier particles immobilized on the substrate was obtained (Comparative Example 3).

In the same manner as in Example 5 except that the purified anti-Candida glabrata DEAE antibody solution was used alone,
A dispersion liquid of antibody-immobilized carrier particles on which abrataDEAE purified antibody was immobilized alone was obtained (Comparative Example 4).

Further, the dispersions of the antibody-immobilized carrier particles obtained in Comparative Examples 3 and 4 were mixed in equal volumes, and the antibody-immobilized carrier particles in which the purified anti-Candida albicans DEAE antibody was immobilized alone and the anti-Candid
A dispersion of antibody-immobilized carrier particles containing both the antibody-immobilized carrier particles on which a glabrata DEAE purified antibody was immobilized alone was obtained (Comparative Example 5).

For Comparative Examples 3 to 5, an agglutination reaction was performed in the same manner as in Example 1. The results obtained are shown in Table 2.

As shown in the sensitivity test results in Table 2, Candida albica
The antibody-immobilized carrier particles of Comparative Example 3, in which an antibody having ns ca-111 as an immunizing antigen was immobilized alone, had a bacterial concentration of 3.1 × 10 ⁵ against Candida albicans ca-111 as the immunizing antigen.
Aggregation was observed with the antigen / ml antigen dilution solution, which was highly sensitive.
For andida glabrata t-g-108 (an antigen corresponding to another antibody), aggregation was only observed with an antigen dilution at a bacterial concentration of 100 × 10 ⁵ cells / ml.

In contrast, the anti-Candida albicans ca-a-
The antibody-immobilized carrier particles in which the anti-Candida glabrata t-g-108 antibody was mixed with 10% of the 111 antibody and the two antibodies were immobilized together had a bacterial concentration of 3.1 × 1 against Candida albicans ca-111.
0 With ⁵ cells / ml antigen dilution Although it showed aggregation and was highly sensitive, Candida glabrata t-
With respect to g-108 (an antigen corresponding to another antibody), aggregation was only observed with an antigen diluent at a bacterial concentration of 100 × 10 ⁵ cells / ml.

In contrast, the anti-Candida albicans ca-a-
The antibody-immobilized carrier particles in which the anti-Candida glabrata t-g-108 antibody was mixed with 10% of the 111 antibody and the two antibodies were immobilized together had a bacterial concentration of 3.1 × 1 against Candida albicans ca-111.
Agglutination was observed at an antigen dilution of ⁵ cells / ml, and as in Comparative Example 3, it was highly sensitive and showed agglutination with Candida glabrata t-g-108 at an antigen dilution of ²⁵ × 10 ⁵ cells / ml. And 4 times higher sensitivity than Comparative Example 3.

Comparative Example 4 in which an antibody using Candida glabrata t-g-108 as an immunizing antigen was immobilized alone, and anti-Candida glabrata t-g-108
A comparison of the antibody-immobilized carrier particles of Example 7 in which the anti-Candida albicans ca-111 antibody was mixed with 10% of the -g-108 antibody and the two antibodies were immobilized together showed that Example 7 was Candida albicans.
bicans ca-111 was 4 times more sensitive than Comparative Example 4.

Further, the antibody-immobilized carrier particles of Comparative Example 5 prepared by mixing Comparative Examples 3 and 4 in equal volumes were Candida albicans ca
-111 and Candida glabrata t-g-108 showed agglutination with an antigen diluent at a bacterial concentration of 6.3 and 50 × 10 ⁵ cells / ml, respectively.

On the other hand, the antibody-immobilized carrier particles in which the two antibodies of Example 5 were immobilized together were Candida albicans ca-111.
In contrast, agglutination was shown with an antigen diluent at a bacterial concentration of 3.1 × 10 ⁵ cells / ml,
The sensitivity is twice as high as that of Comparative Example 5, and Candida glabrata t
Agglutination was observed with an antigen diluent having a bacterial concentration of 12.5 × 10 ⁵ cells / ml against −g-108, and the sensitivity was 4 times higher than that of Comparative Example 5.

The specificity test results in Table 2 show that Candida albicans ca-104 and Candi as antigens of the same species as each immunizing antigen.
Aggregation reactivity with da glabrata t-g-109 was shown.
As shown in Table 2, depending on the antigen, the antibody-immobilized carrier particles of Comparative Examples 3 to 5 showed weak aggregation (+) and (±)
In some cases, it was difficult to determine the presence or absence of aggregation indicated by. On the other hand, the antibody-immobilized carrier particles of the present invention showed clear aggregation (++, ++) for any antigen and were highly sensitive. Next, antigens that differ in species from the respective immunizing antigens but have the same genus, bacteria that are different in genus but are the same fungi, and bacteria that are different in genus, which are usually present in a sample to be measured for a Candida reagent, are so-called normal antigens. Although the agglutination reactivity with bacteria was observed, Examples 5 to 7 had equivalent specificities as compared with Comparative Examples 3 to 5.

(4) Measurement of Patient Samples For each of 10 samples for which candidiasis was determined to be positive by the culture method described in (5-3-1) and 20 samples for which candidiasis was determined to be negative (5-3-2) A) Agglutination reaction was carried out by the method shown in the measurement using antibody-immobilized carrier particles. Aggregation reaction was carried out on the antibody-immobilized carrier particles obtained in Examples 5 to 7 and Comparative Examples 3 to 5 to show clear aggregation (++,
++, +) It was determined that the agglutination reaction was positive. The results are shown in Table 3.

In Table 3, the coincidence rate between the culture method and the agglutination reaction obtained by dividing the number of cases in which the culture method and the agglutination reaction coincided by the total number of the cases was 0.9 or more in Examples 5 to 7 respectively. Showed good agreement with the ratio In Comparative Examples 3 to 5, the coincidence rate was inferior to 0.80 or less. Furthermore, the mismatch rate is inferior to 0.20 or more in Comparative Examples 3 to 5, whereas
In Examples 5 to 7, it was as excellent as 0.10 or less.

As described above in Examples 5 to 7, the Candida reagent using the antibody-immobilized carrier particles of the present invention showed high sensitivity to both Candida albicans and Candida glabrata, which are the causative agents of candidiasis. In contrast, Comparative Examples 3 to
5 was low in sensitivity. In addition, for patient samples, the Candida reagent using the antibody-immobilized carrier particles of the present invention showed a high concordance rate with the culture method and showed sufficient sensitivity and specificity for practical use, whereas the comparative examples 3 to 5 showed a disparity rate. Was inferior to 0.20 or more, which was not suitable for practical use.

Example 8 Candida / trichomonas reagent The anti-Trichomonas antiserum obtained by immunizing rabbits was used for the anti-Candida albicans and anti-Ca
An anti-Trichomonas affinity purified antibody was obtained in the same manner as the affinity purification method for the ndida glabrata antibody.

Anti-Candida albicans, anti-Candida glabrata and anti-Tric
Each affinity purified antibody of homonas (0.25mg /
ml of PB solution) were mixed at a volume ratio of 1: 1 to 1 to obtain an antibody mixture. Next, the average particle diameter is 0.902 μm
Using the polystyrene latex particles of Example 1 as carrier particles, the same procedure as in Example 1 was repeated to carry out anti-Candida albicans, anti-Candida
A dispersion (PB suspension having a particle concentration of about 0.5% by weight) of antibody-immobilized carrier particles, on which both affinity-purified antibodies of glabrata and anti-Trichomonas were immobilized, that is, a Candida / trichomonas detection reagent was obtained.

(2) Aggregation test Three specimens (specimen A, specimen B and specimen C) suspected of having vaginitis were each measured using a detection reagent for Candida and Trichomonas protozoa.

For the detection of Candida bacteria, the vaginal secretion (sample) collected with a cotton swab was placed on chloramphenicol-added Sabouraud glucose agar medium in the same manner as in the section of the measurement by the (5-3-1) culture method in Example 1. After the application, the cells were cultured at 37 ° C. for 48 hours and judged.

For the detection of Trichomonas protozoa, the above sample is placed in a deep portion of a commercially available Trichomonas detection medium (trade name: Nissui, manufactured by Nissui Pharmaceutical Co., Ltd.), cultured at 37 ° C. for 48 hours according to the reagent usage, and then cultured in a mirror. Was inspected and judged.

The determination result of sample A was positive for both Candida and Trichomonas protozoa.

The determination result of sample B was positive for Candida and negative for Trichomonas protozoa.

The determination result of sample C was negative for Candida and positive for Trichomonas protozoa.

An agglutination reaction was carried out using the reagents of Example 8 and Example 1 in the same manner as in Example 1.

As a result of the agglutination test of the sample A, the reagents of Example 8 and Example 1 showed clear aggregation (+). Sample B also showed clear aggregation (+) for both reagents.

Sample C showed clear aggregation (+) with the reagent of Example 8, but did not show aggregation with the reagent of Example 1.

After administering tricomycin to each patient, samples (sample a, sample b, and sample c) were collected again for the purpose of monitoring the progress and measured. Both of the above detection reagents and the reagent of Example 8 and Example 1 showed negative for any sample.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuhiro Miyazaki 2051 Endo, Fujisawa-shi, Kanagawa Prefecture Soda Tokuyama Examiner at Fujisawa Research Institute, Inc. Shoko Yamamura (56) References International Publication 89/5980 (WO, A1)

Claims (2)

(57) [Claims] 1. An immunoagglutination method comprising immobilizing at least two types of antibodies that recognize different antigens and also have cross-reactivity with antigens recognized by other antibodies on the same insoluble carrier particles. Insoluble carrier particles immobilized on antibodies for measurement. 2. An antibody which recognizes at least Candida albicans and has cross-reactivity with Candida glabrata and an antibody which recognizes Candida glabrata and has cross-reactivity with Candida albicans are immobilized on the same insoluble carrier particle. The antibody-immobilized insoluble carrier particles for immunoagglutination measurement according to claim 1.