JP4217516B2 - Method for producing polyclonal antibody against Streptococcus mutans - Google Patents

Description

【００７７】
表１から明らかなように、チオシアン酸ナトリウム溶出により調製したポリクローナル抗体により、１０^５／ｍｌのストレプトコッカス・ミュータンスが検出でき、更に、培養法により得られたストレプトコッカス・ミュータンスの濃度と相関するスポット発色強度が得られた。これらのことから、ここで調製した抗体は、ミュータンスレンサ球菌以外の口腔内レンサ球菌との交差反応性が十分に低く抑えられていることが分かる。
【００７８】
実施例２
［複数のウサギを用いて免疫し、各種カオトロピックイオンを用いて精製して得たポリクローナル抗体の特異性評価］
（１）［ポリクローナル抗体の製造］
実施例１の方法に従って、１０羽のウサギを用いて、菌体懸濁液を免疫することで抗体混合物を調製した。実施例１（Ｂ）ｃ）におけるプロテアーゼ処理菌体でのアフィニティー精製の際に、２Ｍ チオシアン酸ナトリウム、または２Ｍ ヨウ化ナトリウム、または２Ｍ 過塩素酸ナトリウムで溶出し、次いで、プロテインＡ−セファロースで精製した。このようにして各溶出法につき１０種類のポリクローナル抗体を調製した。
【００７９】
（２）［免疫クロマト法ストリップの製造］
実施例１（Ｃ）−Ｉ．記載の方法に従って、上記（１）で調製した３０種類のポリクローナル抗体を使用した免疫クロマト法ストリップを製造した。
【００８０】
（３）［特異性評価試験］
実施例１（Ｃ）−II．と同様の方法により唾液より被検体液を調製し、上記（２）で作製した免疫クロマト法ストリップにより分析した。チオシアン酸ナトリウムにより溶出した抗体の結果を表２に、
【００８１】
【表２】

【００８２】
【表３】

【００８３】
ヨウ化ナトリウムにより溶出した抗体の結果を表３に、
【００８４】
【表４】

【００８５】
【表５】

【００８６】
過塩素酸ナトリウムにより溶出した抗体の結果を表４に示す。
【００８７】
【表６】

【００８８】
【表７】

【００８９】
表２〜４に示したように、チオシアン酸ナトリウム、ヨウ化ナトリウム、過塩素酸ナトリウムで溶出した場合、１０種類のすべての抗体で、１０^５／ｍｌのストレプトコッカス・ミュータンスが検出でき、更に、培養法により得られたストレプトコッカス・ミュータンスの濃度と相関するスポット発色強度が得られた。ウサギ個体に関係なく、ミュータンスレンサ球菌以外の口腔内レンサ球菌との交差反応性が十分に低く抑えられた抗体が安定に得られた。
【００９０】
比較例１
［複数のウサギを用い、酸溶出法により精製して得たポリクローナル抗体の特異性評価］
（１）［ポリクローナル抗体の製造］
実施例２（１）で得られた各抗体混合物を用いて、酸溶出法によるアフィニティー精製を行った。
【００９１】
実施例１（Ａ）の方法に従ってプロテアーゼ処理菌体を調製した。該プロテアーゼ処理菌体をＰＢＳで３回洗浄し、次いで０．１Ｍ グリシン塩酸緩衝液（ｐＨ２．０）で３回洗浄し、更にＰＢＳで３回洗浄し、プロテアーゼ処理菌体懸濁液（Ａ_６００＝１２．５）を調製した。
【００９２】
次いで、実施例１（Ｂ）ｃ）と同様の方法により抗体混合物とプロテアーゼ処理菌体懸濁液とを混合し、ＰＢＳで３回洗浄した後、０．５ｍｌの０．１Ｍ グリシン塩酸緩衝液（ｐＨ２．０）に菌体を懸濁して、吸着していた抗体を溶出し、遠心分離により上清を回収し、１Ｍトリス−塩酸（ｐＨ９．０）を添加しｐＨ７．４に調整した。同様の溶出操作を４回行い、各画分のタンパク質量を２８０ｎｍの吸光度により測定した。
【００９３】
次いで、あらかじめＰＢＳで平衡化した１ｍｌのプロテインＡ−セファロース（アマシャムファルマシアバイオテク社）を充填したカラムに上記溶出液を添加し、実施例１（Ｂ）と同様の方法により抗体を溶出し、１０種類のポリクローナル抗体を調製した。
【００９４】
（２）［免疫クロマト法ストリップの製造］
実施例１（Ｃ）−Ｉ．記載の方法に従って、上記（１）で調製したポリクローナル抗体を使用した免疫クロマト法ストリップを製造した。
【００９５】
（３）［特異性評価試験］
実施例３（３）で調製した抗原抽出液を、上記（２）で作製した免疫クロマト法ストリップにより分析した。結果を表５に示す。
【００９６】
【表８】

【００９７】
【表９】

【００９８】
表５に示したように、酸溶出法によるアフィニティー精製でポリクローナル抗体を製造すると、ウサギによっては非特異的反応が検出される場合があることが明らかとなった。
【００９９】
【発明の効果】
本発明によれば、ミュータンスレンサ球菌以外の口腔内レンサ球菌との交差反応性を十分に低く抑えたストレプトコッカス・ミュータンスに特異的なポリクローナル抗体を、免疫動物個体差に影響されることなく安定して製造することが可能となる。
【図面の簡単な説明】
【図１】本図は、本発明の免疫クロマト法で使用するストリップの各部材の概略図である。
【図２】本図は、本発明の免疫クロマト法で使用するストリップの側面図である。
【符号の説明】
１・・・ストリップ
２・・・サンプルパッド
３・・・コンジュゲートパッド
４・・・展開メンブレン
５・・・吸収パッド
６・・・検出ライン
７・・・コントロール判定ライン[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a polyclonal antibody specific for Streptococcus mutans.
[0002]
[Prior art]
In general, it is known that a group of lactic acid-fermenting bacteria called mutans streptococci are deeply involved in caries development.
[0003]
These mutans streptococci include Streptococcus cricetus (S. critus, serotype a), Streptococcus lattus (S. rattus, serotype b), Streptococcus mutans (S. mutans, serotype c, e, f). ), Streptococcus fels (S. ferus, serotype c), Streptococcus macaque (S. macacae, serotype c), Streptococcus sobrinus (S. sobrinus, serotype d, g), Streptococcus dounei (S. downney) The serotype h) is classified into seven types that are serologically and genetically different.
[0004]
Conventionally, the concentration of these mutans streptococci in saliva is 10⁵-10⁶In the case of pcs / ml, there is a risk of caries. 10⁶It is said that the number of cells / ml or more is particularly dangerous. By knowing the abundance of mutans streptococci in the oral cavity of a person, it is possible to determine the degree of caries risk of the person. In general, the concentration of these mutans streptococci is measured by selectively culturing mutans streptococci in saliva using a medium containing bacitracin and examining the number of colonies (a measurement kit for that is also commercially available). Have been). And the density | concentration of each mutans streptococcus in saliva can be known once by identifying the colony of each microbe from the colonies obtained by culture | cultivating similarly, and investigating the number. Known identification methods include biochemical methods such as sugar fermentation tests, genetic methods using DNA probes, immunological methods using serotype-specific antibodies, and the like.
[0005]
In recent years, it has been clarified that two species of Streptococcus mutans and Streptococcus sobrinus exist in the human oral cavity among mutans streptococci. In particular, Streptococcus mutans was isolated from the human oral cavity at a high frequency (separated from more than 90% of people) and was found to be deeply related to the occurrence of caries.
[0006]
Currently, a culture method is widely used as a method for measuring mutans streptococci in the oral cavity. However, in the culture method, the culture operation is indispensable, and further, skilled techniques are required to identify the bacterial species from the form of the isolated colonies, so that there are problems in terms of inspection time and complexity of operation. is there.
[0007]
As a method for directly detecting Streptococcus mutans from clinical specimens such as plaque and saliva, immunological measurement methods using various monoclonal antibodies and polyclonal antibodies have been reported. These methods have the advantage that the time required for detection can be greatly shortened without the need to culture bacteria.
[0008]
It is known that streptococci classified into various genus Streptococcus are resident in the oral cavity. Although the presence ratio in the bacterial flora varies depending on each site in the oral cavity, streptococcus belonging to the genus Streptococcus is known to occupy nearly 50% of the bacterial flora in the plaque and saliva used to determine caries risk. Cage (for example, Non-Patent Document 1), and the total number of bacteria in 1 ml of saliva is 10⁸It is said to be more than pieces / ml. The number of mutans streptococci is generally 10⁷It can be said that most streptococci in the oral cavity are streptococci other than mutans streptococci because the number is less than one.
[0009]
Therefore, for the determination of caries risk, 10⁸10 / ml mixed in oral streptococci other than mutans streptococci⁵It is necessary to measure Streptococcus mutans on the order of individual / ml, that is, even if Streptococcus in the oral cavity other than Streptococcus mutans is about 1000 times the amount of Streptococcus mutans. There is. For such measurements, 10⁵Compared with the reactivity with Streptococcus mutans / ml / ml⁸An antibody with a high titer and a very high specificity is required that has a very low cross-reactivity with oral streptococci other than 1 / ml mutans streptococci.
[0010]
As a method using a monoclonal antibody, the enzyme-labeled monoclonal antibody against Streptococcus mutans is mixed with a specimen and filtered through a membrane filter having a pore size of 0.45 μm to filter only the enzyme-labeled antibody bound to Streptococcus mutans. A method of detecting by color development after addition of an enzyme substrate has been proposed (Non-patent Document 2, Patent Document 1), and 10 monoclonal antibodies are used in the method.⁵10 levels of Streptococcus mutans reactivity and 10⁷Cross-reactivity with individual levels of Streptococcus salivarius is comparable. 10 in the oral cavity⁷-10⁸There may be a single level of Streptococcus salivarius. In this case, there is a problem that Streptococcus mutans cannot be measured accurately.
[0011]
Focusing on the reactivity with oral streptococci other than mutans streptococci, a measurement method using a monoclonal antibody against Streptococcus mutans that does not react with Streptococcus mitis, Streptococcus salivarius, Streptococcus sangis has been proposed ( Patent Document 2). The specificity of the monoclonal antibody is constant (10⁸The reactivity of the antibodies against the above three bacterial species and Streptococcus mutans cultures was examined by a 2-fold dilution series of the antibodies, and the volume-action curve of the antigen-antibody reaction was plateau It is disclosed that the cross-reactivity to Streptococcus mitis, Streptococcus salivarius and Streptococcus sangis is not detected even in an excessive antibody range. But 10⁵The reactivity to Streptococcus mutans has not been investigated, and Streptococcus mutans is accurately detected when oral streptococci other than mutans streptococci are present in an amount about 1000 times that of Streptococcus mutans. Whether it can be measured is unknown. In addition, there is a problem that the reactivity of other oral streptococci with Streptococcus gordonii and Streptococcus oralis has not been studied at all.
[0012]
  Also, it does not react with Streptococcus mittis, Streptococcus salivarius, Streptococcus sangis and Porphyromonas gingivalis, and Streptococcus mutans and Streptococcus sobrinusreactA measurement method using a monoclonal antibody has been proposed (Patent Document 3). When the reactivity of the monoclonal antibody to a certain amount of culture (dry weight 5 μg) was examined, Streptococcus sangis, Streptococcus salivarius, Streptococcus mitis, Streptococcus lattus, Streptococcus fels, Streptococcus anginasus, Lactobacillus zeus , No cross-reactivity to Porphyromonas gingivalis is detected, and 10⁵Per ml of Streptococcus mutans can be measured,⁶Disclosed is the property that no cross-reactivity to Streptococcus salivarius / ml / ml is detected. However, in this case as well, it is unclear whether Streptococcus mutans can be accurately measured when the oral streptococci other than mutans streptococci are present in an amount about 1000 times that of Streptococcus mutans, There is a problem that the reactivity of other oral streptococci with Streptococcus gordonii and Streptococcus oralis has not been studied at all.
[0013]
On the other hand, as a method using a polyclonal antibody against Streptococcus mutans, a method using a latex agglutination method on a glass slide has been reported (Non-patent Documents 3 and 4, Patent Document 4). In general, Streptococcus mutans is not very antigenic, and it is known that it is difficult to obtain a highly specific polyclonal antibody even if the antibody causing the cross reaction is removed by affinity purification. However, in this method, a polyclonal antibody prepared by immunizing a rabbit with Streptococcus mutans cells is used without affinity purification. Therefore, it is considered that the specificity of the polyclonal antibody used here is low. Therefore, in this method, 1 × 10⁶A positive reaction was obtained only in the bacterial cell sample solution having a concentration of not less than 1 cell / ml, and sufficient sensitivity was not achieved.
[0014]
Thus, regarding the measurement of Streptococcus mutans in clinical specimens prepared directly from saliva or dental plaque, the detection limit is 10 for the determination of the caries risk.⁵Although a simple measurement method per unit / ml is desired, a Streptococcus mutans measurement method that satisfies this requirement has not been known.
[0015]
The present inventors consider that there is an effect of oral streptococci other than mutans streptococci present in the sample liquid as one of the causes that detection sensitivity does not increase, and as a result of earnest examination, it exists in the oral cavity Previously proposed a polyclonal antibody capable of specifically detecting and quantifying Streptococcus mutans, and a method for producing the same, with sufficiently low cross-reactivity to oral streptococci other than mutans streptococci (Japanese Patent Application No. 2001-384595). This production method is carried out by immunizing an immunized animal with a microbial cell or an antigen extract from the microbial cell as an immunogen to obtain an antibody mixture, and then using the immunogen as an affinity carrier and performing affinity purification. The At this time, at least one of the immunogen for the immunized animal and the antigen used for affinity purification, the cell surface protein considered to be a substance causing the cross-reaction existing on the surface of Streptococcus mutans cells was removed by protease treatment. Preparation of antibodies capable of specifically detecting Streptococcus mutans in clinical specimens prepared directly from saliva or plaque by using bacterial cells (hereinafter also referred to as “protease-treated bacterial cells”) or antigen extracts thereof It becomes possible to do.
[0016]
Here, affinity purification involves contacting the antibody mixture with an affinity carrier, adsorbing an antibody that reacts only with Streptococcus mutans (hereinafter also referred to as “specific antibody”) to the affinity carrier, and washing the affinity carrier. After removing the substance, the specific antibody is eluted from the affinity carrier. In general, since the binding force of the antigen-antibody complex is strong, severe conditions such as acidic conditions, alkaline conditions, use of denaturing agents, etc. are applied for elution of the antibody from the affinity carrier. It is often performed (for example, Non-Patent Document 6).
[0017]
[Non-Patent Document 1]
Supervised by Hidehiko Takekasa, "Molecular biology of caries bacteria-Research results and prospects", 1st edition, Quintessence Publishing, 1997, p29-37
[Non-Patent Document 2]
Yutaka Yasutomi et al., "Journal of Pediatric Dentistry", Volume 30, 1992, p186-193
[Patent Document 1]
Japanese Patent No. 3093833
[Patent Document 2]
JP-A-10-36400
[Patent Document 3]
JP 2001-172299 A
[Non-Patent Document 3]
Takei Tsutomu, "Osaka University Medical Journal", Volume 35, 1990, p93-109
[Non-Patent Document 4]
Takei, “T. Archs. Oral. Biol.”, 37, 1990, p99-104.
[Patent Document 4]
Japanese Patent Laid-Open No. 1-250067
[Non-Patent Document 5]
Edited by Shigeyuki Hamada, "Caries and periodontal disease", Nippon Dental Detailed Co., Volume 1, 1982, p17-23
[Non-Patent Document 6]
"Affinity Chromatography Handbook", Amersham Pharmacia Biotech, 1999
[0018]
[Problems to be solved by the invention]
However, in the method for producing a polyclonal antibody specific for Streptococcus mutans, when elution of the antibody from the affinity carrier is carried out by making the eluate acidic, due to individual differences among immunized animals, It has been found that the variation in cross-reactivity of the produced polyclonal antibody with oral streptococci other than mutans streptococci increases, and there are cases where antibodies are produced in which this cross-reaction cannot be suppressed sufficiently low. This is to reduce the productivity of the target antibody in the case where an antibody mixture is obtained from a large number of immunized animals by the above-described method and the specific antibody is produced in a large amount. It was a big obstacle to doing.
[0019]
Therefore, the present invention is specific to Streptococcus mutans, which is stable without being affected by individual differences in immunized animals, and has sufficiently low cross-reactivity with oral streptococci other than mutans streptococci. It aims at providing the method of manufacturing a polyclonal antibody.
[0020]
[Means for Solving the Problems]
As a result of intensive studies by the present inventors in view of the above-mentioned problems, the inventors have found a method capable of stably producing a polyclonal antibody specific to Streptococcus mutans without being affected by individual differences among immunized animals, and completed the present invention. It came to do.
[0021]
  That is, the present invention provides a non-human immunized animal with a) a whole cell of Streptococcus mutans obtained by removing a bacterial cell surface protein by protease treatment,Orb) A polyclonal antibody production method comprising immunizing whole cells of Streptococcus mutans to obtain an antibody mixture, and then purifying the antibody mixture by affinity purification method,
A) When the antigen to be used for immunization to the non-human immunized animal is a), the antigen used as a carrier in the affinity purification method is a whole strain of Streptococcus mutans from which the cell surface protein has been removed by protease treatment. Body, concernedProtease treatmentAn antigen extract of bacterial cells, a whole cell of Streptococcus mutans, and at least one selected from antigen extracts of the bacterial cells,
B) When the antigen to be used for immunization to non-human immunized animals is b), the antigen used as a carrier in the affinity purification method is a whole strain of Streptococcus mutans from which cell surface proteins have been removed by protease treatment. Body or concernedProtease treatmentAn antigen extract of bacterial cells,
A method for producing a polyclonal antibody against Streptococcus mutans, wherein chaotropic ions are used for elution of the antibody from the carrier in the affinity purification.
[0022]
By adapting the production method of the present invention, for example, the reactivity to Streptococcus mutans described in Japanese Patent Application No. 2001-384595 is such that Streptococcus gordonii, Streptococcus salvarius, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguisu. It is possible to easily and stably produce a polyclonal antibody characterized by being 1000 times or more compared to the reactivity to. The inventors of the present invention carefully examined the immunization method for producing a specific antibody against the above-mentioned Streptococcus mutans, elution conditions of the antibody from the affinity carrier, etc., and found an optimal combination of these conditions. It was made by being issued.
[0023]
DETAILED DESCRIPTION OF THE INVENTION
In short, the method for producing a polyclonal antibody according to the present invention comprises:
(1) A non-human immunized animal is immunized with a whole cell of Streptococcus mutans from which bacterial cell surface protein has been removed by protease treatment to obtain an antibody mixture, and then a) bacterial cell surface protein by protease treatment. Streptococcus mutans cells removed from theProtease treatmentA specific antibody is recovered by an affinity purification method using at least one selected from an antigen extract of a bacterial cell, a whole cell of Streptococcus mutans, and an antigen extract of the bacterial cell as an antigen, or
(2) A non-human immunized animal is immunized with b) whole cells of Streptococcus mutans to obtain an antibody mixture, and then b) whole cells of Streptococcus mutans from which cell surface proteins have been removed by protease treatment. Or the relevantProtease treatmentRecover specific antibody by affinity purification using antigen extract of bacterial cells as antigen
Applied in the method.
[0024]
  In these production methods, Streptococcus mutans protease-treated bacterial cells are used as at least one of an immunogen for an immunized animal and an antigen used for affinity purification.(In the case of an antigen used for affinity purification, an antigen extract of the Streptococcus mutans protease-treated cells may be used.)is doing. In the Streptococcus mutans protease-treated cells, the cell surface proteins considered to be substances that cause the cross-reaction existing on the surface of the cells are removed by the action of the protease.
[0025]
  Therefore, such protease-treated bacteriabodyCan be used as an immunogen for immunized animals, production of antibodies that cause a cross-reaction can be greatly suppressed, and highly specific polyclonal antibodies can be produced. On the other hand, if the protease-treated bacterial cells or antigen extracts thereof are used for affinity purification, these antigens have been removed from the bacterial cell surface proteins that cause cross-reactions existing on the bacterial cell surface as described above. The antibody that causes the corresponding cross-reaction contained in the antibody mixture is not adsorbed to the affinity carrier, and as a result, a highly specific polyclonal antibody can be produced.
[0026]
In the present invention, Streptococcus mutans means a microbial cell whose serotype is classified into c, e, and f types in the mutans streptococci group. As a standard strain of serotype c, strains such as Ingbritt and MT6R, as a standard strain of serotype e type, strains such as LM7 and P4, and as a standard strain of serotype f type, strains such as SE11 and OMZ175 Each is illustrated.
[0027]
The polyclonal antibody (hereinafter also simply referred to as “antibody”) is an antibody fraction contained in the antiserum, and the globulin class of the antibody is not limited, and any currently known globulin class. Are also included. The meaning includes not only normal antibody molecules but also partial degradation products (Fab, Fab ′, Fab′2, etc.) of the antibody, and partial structures in which the active fragments (antigen recognition sites of the antibody) are present. It is.
[0028]
  In the production method of the present invention, as an immunogen for an immunized animal, all of the above-mentioned Streptococcus mutans serotypes c, e, and f, or those three types of Streptococcus mutans protease-treated cells are used. Can be used. theseAre used as whole cells.Each immunogen derived from each serotype of Streptococcus mutans (total cells,OrProtease-treated bacteriabody) The immunized animal can be immunized alone, but they may be mixed and immunized.
[0029]
As the total cells of Streptococcus mutans, dead cells that have been subjected to pretreatment such as formalin treatment or heat treatment, cells that have been cryopreserved, and the like can be used.
[0030]
Protease-treated cells can be prepared by treating the entire cells with protease. As the protease, various known proteases or a mixture thereof can be used without limitation, and examples thereof include pronase, proteinase K, pepsin, trypsin, chymotrypsin and the like.
[0031]
In the protease treatment method, for example, the whole cell is suspended in a buffer solution or the like prepared so as to be close to the optimum pH of the reaction of the protease to be used, and the protease is added and mixed at 15-50 ° C. for 10 minutes to 120 with good mixing. This can be done by keeping the temperature warm. After completion of the reaction, for example, the reaction solution is centrifuged or filtered to remove the protease and collect the protease-treated cells. In order to avoid contamination with protease, the cells collected by centrifugation or filtration are suspended in a buffer solution such as phosphate physiological saline buffer (pH 7.4) (hereinafter sometimes abbreviated as “PBS”). It is preferable to perform the washing operation 3 to 5 times by collecting the cells by the same operation.
[0033]
These immunogens can be used for immunization as they are, or can be used in combination with a carrier for immunization. As carriers, mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, polyalanyl lysine, dipalmityl lysine, tetanus toxoid or polysaccharide Conventionally known carriers such as these can be suitably used.
[0034]
  As an immunogen for producing the antibody of the present invention, the whole antibody of Streptococcus mutans or protease-treated bacteria can be directly immunized because of the ease of production and the high titer of the antibody obtained.Use. Furthermore, the method of directly immunizing all cells of Streptococcus mutans has an advantage that high-titer antibodies can be stably obtained because the antigenicity of all cells is higher than that of protease-treated cells.
[0035]
As the animal for immunizing the immunogen as described above, animals generally used for obtaining antibodies can be used, but mammals such as mice, goats, rabbits, and guinea pigs are preferably used. .
[0036]
As a method for immunizing these animals with an immunogen, a conventionally known method can be adopted without limitation, and for example, it can be carried out by injecting the immunogen subcutaneously or intravenously. The immunogen is, for example, a fungusBodyMay be injected directly, or fungusTo the bodyAn adjuvant may be added and mixed for injection. For example, when directly immunizing bacterial cells, it is also possible to use a suspension of bacterial cells obtained after culturing Streptococcus mutans in a brain heart infusion (hereinafter abbreviated as BHI) liquid medium or the like. . As the adjuvant, conventionally known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and pertussis adjuvant can be suitably used.
[0037]
After immunizing these animals with an immunogen, blood is collected and serum is prepared to obtain a mixture of various antibodies including a polyclonal antibody against Streptococcus mutans, that is, an antibody mixture.
[0038]
In this antibody mixture, there are antibodies that react simultaneously with oral streptococci other than mutans streptococci, so specific antibodies are selectively recovered from the antibody mixture.
[0039]
  In the present invention, an affinity purification method is employed as a method for purifying a specific antibody from an antibody mixture. As an antigen (antigen-carrying substance) used in this affinity purification method, at least one selected from all Streptococcus mutans cells, protease-treated cells, and antigen extracts of these cells is used. However, the immunogen to the immunized animal is Streptococcus mutans whole bacteriaIn the bodyIn such a case, the antigen used in the affinity purification method is specifically used as a protease-treated cell or its antigen extract.Here, the extraction of antigens from all cells of Streptococcus mutans or protease-treated cells is not particularly limited, but proteins, tycoic acid, etc. present on the surface of the cells are other oral streptococci. As there are many things with high homology with It is preferred to extract the polysaccharide antigen present in Polysaccharide antigens can be extracted by heating the cell suspension (Rants, LA, Stanford. Med. Bull. 13: 290-291, 1955.) or extracting with nitrous acid (Takei Tsutomu. Osaka University Medical Journal. 35: 93-109, 1990.). The extracted polysaccharide antigen can be used as an antigen as it is, or can be used after further purification.
[0040]
The affinity purification operation is performed by bringing an insoluble carrier on which the antigen is immobilized (also referred to as “antigen-immobilized carrier”) and an antibody mixture into contact with each other, adsorbing a specific antibody in the antibody mixture, and then immobilizing the antigen-immobilized carrier. Can be performed by removing contaminants by washing with a buffer solution or the like, and then eluting the specific antibody from the antigen-immobilized carrier. In addition, whole cells of Streptococcus mutans or protease-treated cells are used as they are without using an insoluble carrier, and contacted with an antibody mixture, thereby adsorbing specific antibodies to these cells and separating them, Next, the specific antibody may be eluted from these cells. The latter method is more preferable because it is easy to operate and can simultaneously collect multiple types of antibodies that react with multiple types of antigens present on the surface of Streptococcus mutans cells. is there.
[0041]
In this affinity purification method, the present invention has a great feature in that chaotropic ions are used when a specific antibody is eluted from an antigen-immobilized carrier. Thereby, the specificity of the polyclonal antibody obtained by elution is further improved, and its excellent properties are hardly affected by individual differences among immunized animals. Therefore, a highly specific polyclonal antibody can be produced with stable quality.
[0042]
Here, chaotropic ions are a general term for ions that have the effect of breaking the cage structure of water molecules in an aqueous solution. By adding these ions, reduction in surface tension, increase in hydration entropy in the vicinity of ions, etc. are observed. The Chaotropic ions affect antigen-antibody reactions by increasing the water solubility of hydrophobic molecules and weakening hydrophobic bonds. As such chaotropic ions, known ones can be used without particular limitation. Specific examples of chaotropic ions include thiocyanate ions, iodine ions, perchlorate ions, trichloroacetate ions, bromine ions, nitrite ions, and the like. Among these, thiocyanate ion, iodine ion, and perchlorate ion are particularly preferable because they have a small effect on the antibody and can be purified stably.
[0043]
These chaotropic ions are suitably used, for example, as an eluent in the range of 0.5 to 4M, and one kind of chaotropic ion may be used alone, or a plurality of chaotropic ions may be used in combination.
[0044]
The eluate containing chaotropic ions may be prepared by dissolving a salt of chaotropic ions in water, or, for example, by dissolving in a buffer solution such as a phosphate buffer or Tris-HCl buffer to elute the desired pH. A liquid can also be prepared. As the salt of chaotropic ion, for example, sodium salt, potassium salt, ammonium salt, etc. of each chaotropic ion can be suitably used.
[0045]
In the method for affinity purification using an antigen-immobilized carrier, the insoluble carrier is any conventionally known carrier such as agarose, dextran, cellulose, polyacrylamide, polystyrene, vinyl chloride, glass, silicone rubber, or porous silica beads. Can be used without limitation.
[0046]
In addition, as a method for immobilizing the antigen on these insoluble carriers, a chemical binding method such as cyanogen bromide activation method and a method utilizing physicochemical adsorption can be used without any limitation.
[0047]
As a method for contacting and separating the antigen-immobilized carrier and the antibody mixture in the affinity purification method, any of a batch method and a chromatography method can be applied. It is also possible to carry out a combination of both methods.
[0048]
In the chromatographic method, the antigen-immobilized carrier is packed into the column, the antibody mixture is added to the column, then a buffer solution or the like is added to the column, the fraction that is not adsorbed on the column is discarded, and then the eluate is removed. What is necessary is just to collect | recover the antibody which added to the column and adsorb | sucked. In the batch method, a solution in which an antigen-immobilized carrier is suspended is mixed with an antibody mixture, and after sufficiently allowing an antigen-antibody reaction, a precipitate is collected by, for example, centrifugation, and the precipitate is suspended in a buffer solution or the like. The antibody may be eluted by washing by repeating the operation of collecting the precipitate by turbidity and repeating the treatment a plurality of times, and finally suspending the precipitate in the eluate.
[0049]
The contact and separation method with the antibody mixture when using Streptococcus mutans whole cells or protease-treated cells as they are, is the same as the above batch method, that is, the cell suspension and the antibody mixture are mixed, After sufficient antigen-antibody reaction, the precipitate can be collected by, for example, centrifugation.
[0050]
According to the method for producing an antibody of the present invention described above, a polyclonal antibody having high specificity and reactivity against Streptococcus mutans can be stably produced regardless of individual differences among immunized animals. This polyclonal antibody usually has a reactivity with Streptococcus mutans that is 1000 times or more compared with the reactivity with Streptococcus gordonii, Streptococcus salivarius, Streptococcus oralis, Streptococcus mittis, and Streptococcus sangis. Thus, if a polyclonal antibody having high specificity for Streptococcus mutans is applied to an immunoassay, even if other streptococci are present in the sample liquid, 10⁵Streptococcus mutans at a concentration of about 1 / ml can be detected.
[0051]
The sample liquid used for this immunological measurement is not particularly limited as long as it contains Streptococcus mutans. For example, saliva, plaque, bacterial cell culture medium, cultured bacterial cell suspension, etc. are used. it can. In particular, body fluids including saliva and plaque are preferably used as specimens for examining caries risk. Note that saliva and dental plaque may be contained alone in the subject liquid or as a mixture. In addition, the sample liquid is treated by a conventionally known method such as sonication for the purpose of dispersing insoluble matters contained in the sample liquid, mixing with a crushing agent such as glass beads, and stirring, and then subjected to analysis. You can also. In addition, an extract prepared by subjecting the above-described sample liquid to an extraction process can also be measured as the sample liquid. Examples of antigen extraction methods include, for example, a heat treatment method (Rants, LA, Stanford. Med. Bull. 13: 290-291, 1955.), nitrous acid, alkali, salt, chelating agent, chaotropic ion, Extraction method using a surfactant (Takei Tsutomu. Osaka University Medical Journal. 35: 93-109, 1990), treatment method with an enzyme that destroys the membrane structure of bacterial cells (Japanese Patent Laid-Open No. 9-178752) Etc.) can be used without limitation.
[0052]
The immunological measurement method is not particularly limited, and conventionally known immunity methods such as immunoagglutination method, optical immunoassay method, labeled immunoassay method, and combinations thereof, which are known as methods for measuring antigen by antigen-antibody reaction The measurement method can be used without limitation. The measuring reagent for carrying out these immunological measuring methods is not particularly limited as long as it is an immunological measuring reagent containing a polyclonal antibody obtained by the present invention, and a solution containing the antibody of the present invention; The antibody of the invention immobilized on an insoluble carrier such as a particle or a membrane, or a suspension thereof; a labeling substance such as a radioactive substance, enzyme, various dyes, colloids, or various colored particles bound to the antibody of the present invention Or their solutions or suspensions; or such labeled antibodies of the present invention immobilized on insoluble carriers such as particles or membranes; or suspensions thereof; and combinations thereof. Can take a variety of forms.
[0053]
Among various immunological measurement methods, the latex quantification method and enzyme immunoassay method can process a large number of sample liquids using an automatic analyzer, making it suitable as a test method for mass screening and the like. It is. In addition to simplicity, flow-through immunoassay, immunochromatography, and latex agglutination methods are capable of rapid measurement without the need for special knowledge or equipment, and can also be implemented in dental clinics and homes. Therefore, it is suitable as a general-purpose inspection method.
[0054]
A more suitable measurement method is an immunochromatography method, and in particular, a sample pad for temporarily absorbing and retaining a sample liquid as described in Japanese Patent Application No. 2001-384595, and a labeled antibody are temporarily incorporated. Conjugate pad for holding and detection antibody immobilized, analyte liquid temporarily absorbed and retained in the sample pad, and labeled antibody accompanying the analyte liquid and flowing out of the conjugate pad A method using an immunochromatographic test strip in which a development membrane on which the membrane is developed is joined in this order is optimal. In the immunochromatographic test strip, the polyclonal antibody produced in the present invention is used as one or both of a labeled antibody and a detection antibody. Details for carrying out these various immunological measurement methods are described in Japanese Patent Application No. 2001-384595, as well as Japanese Patent Application Laid-Open No. 2001-302697 and the like. Just do it.
[0055]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by a following example.
[0056]
Example 1
[Production and evaluation of polyclonal antibodies by thiocyanate elution method]
(A) [Production of antibody mixture against Streptococcus mutans]
3.7 g of BHI (DIFCO) was dissolved in 100 ml of ultrapure water and then autoclaved to prepare a BHI liquid medium. Ingbritt was incubated at 37 ° C. for 5 hours in 100 ml of BHI liquid medium under anaerobic conditions (N₂: H₂: CO₂= 80:10:10). The culture solution was centrifuged at 4000 g for 5 minutes to remove the culture medium component of the supernatant and collect the bacterial cell precipitate. Next, the precipitate was suspended in 100 ml of PBS and subjected to the same centrifugation three times to wash the precipitate. The cell pellet is suspended in PBS and A₆₀₀= 1.0, and a cell suspension of Ingbritt was prepared.
[0057]
Separately from the above, the Ingbritt cell precipitate obtained by culturing in BHI medium was washed, suspended in 0.1 M Tris-HCl buffer (pH 8.0), and A₆₀₀= 15. Pronase (Wako Pure Chemical Industries, Ltd.) was added to this so that it might become 5 mg / ml, and it heat-retained at 37 degreeC for 1 hour. After completion of the reaction, the mixture was centrifuged to collect the cell precipitate. Subsequently, the precipitate was suspended in 10 ml of PBS and subjected to the same centrifugation three times to wash the precipitate. The cell pellet is suspended in PBS and A₆₀₀= 1.0, and a protease-treated cell suspension of Ingbritt was prepared.
[0058]
Immunization was performed as follows.
[0059]
0.5 ml of the bacterial cell suspension or protease-treated cell suspension and 0.5 ml of Freund's complete adjuvant were mixed well and injected subcutaneously into rabbits. Two weeks later, 1 ml of the bacterial cell suspension or protease-treated bacterial cell suspension and 1 ml of Freund's incomplete adjuvant were mixed well and boosted subcutaneously into the rabbit. Similar booster immunizations were performed at 4th and 6th weeks. After confirming the increase in the titer by the degree of the agglutination reaction of the bacterial cells using a slide glass, blood was collected according to a standard method 2-3 weeks after the final immunization, and Streptococcus An antibody mixture against mutans (an antibody mixture obtained by immunizing a cell suspension) was obtained.
[0060]
Separately from this, even when 0.5 ml of protease-treated cell suspension was used, the same immunization was performed, and an antibody mixture against Streptococcus mutans (obtained by immunizing the protease-treated cell suspension) Antibody mixture).
(B) [Purification of polyclonal antibody against Streptococcus mutans]
According to the method of (A), Ingbritt cultured in 1 L of BHI liquid medium or protease-treated Ingbritt was washed 3 times with PBS, then washed 3 times with 2M sodium thiocyanate, and further washed 3 times with PBS. Suspension and protease-treated cell suspension (A₆₀₀= 12.5) was prepared.
[0061]
Then
a) Mixing this protease-treated cell suspension with 0.5 ml of the antibody mixture obtained by immunizing the protease-treated cell suspension prepared in (A),
b) mixing the above cell suspension with 0.5 ml of the antibody mixture obtained by immunizing the protease-treated cell suspension prepared in (A), and
c) Mixing the protease-treated cell suspension with 0.5 ml of the antibody mixture obtained by immunizing the cell suspension prepared in (A).
Each operation was performed, and each liquid mixture was reacted at 4 ° C. for 60 minutes. Each mixed solution was centrifuged at 4000 g for 5 minutes to recover each bacterial cell. The cells were suspended in 10 ml of PBS and washed by performing the same centrifugation operation three times.
[0062]
Next, each bacterial cell was suspended in 0.5 ml of 2M sodium thiocyanate solution, the adsorbed antibody was eluted, and the supernatant was collected by centrifugation. The same elution operation was performed again, and the protein amount of each fraction was measured by absorbance at 280 nm.
[0063]
Next, PBS was added to each of the above eluates to a column packed with 1 ml of protein A-Sepharose (Amersham Pharmacia Biotech) pre-equilibrated with PBS. The column was washed with 5 ml of PBS and eluted with 5 ml of 0.1 M glycine-hydrochloric acid buffer (pH 3.0). Immediately after addition of 1 M Tris-hydrochloric acid (pH 9.0), the pH was adjusted to 7.4. It was adjusted. The eluted fraction of IgG is A₂₈₀Each was confirmed by measuring.
[0064]
As described above, a) a polyclonal antibody “M-1” obtained by purifying an antibody mixture obtained by using a protease-treated cell suspension as an antigen with a protease-treated cell was obtained. In addition, b) a polyclonal antibody “M-2” was obtained by purifying an antibody mixture obtained by using a protease-treated cell suspension as an antigen with the cells. Furthermore, c) A polyclonal antibody “M-3” was obtained by purifying the antibody mixture obtained using the cell suspension as an antigen with protease-treated cells.
(C) [Specificity evaluation of polyclonal antibody]
I. [Preparation of strip for immunochromatography for measuring Streptococcus mutans]
(1) [Preparation of polyclonal antibody against Streptococcus mutans labeled with colloidal gold]
100 mM K in 10 ml of commercial gold colloid solution (British BioCell International) with colloid particle size of 40 nm₂CO₃Was added to the solution to adjust the pH to 9.0, followed by 0.22 μm filter treatment. When the absorbance at 520 nm of the colloidal gold solution was measured, A₅₂₀= 1.0.
[0065]
Next, 64 μl of a 2 mM borate buffer solution (pH 9.0) of each polyclonal antibody of M-1, M-2, and M-3 adjusted to 1 mg / ml was added to the gold colloid solution with stirring. Left for 5 minutes. Next, 1.1 ml of 10% skim milk-2 mM borate buffer (pH 9.0) was added with stirring (final concentration of skim milk 1%) and left at room temperature for 30 minutes. Next, the reaction solution was centrifuged at 10 ° C., 10000 g for 30 minutes, and after removing the supernatant, 2 ml of 2 mM PBS (pH 7.4) was added to resuspend the lower gold colloid fraction. When the absorbance at 520 nm of the resuspended fraction was measured, A₅₂₀= 4.9.
[0066]
The gold colloid fraction of each antibody obtained (hereinafter sometimes referred to as “gold colloid label M-1”, “gold colloid label M-2”, and “gold colloid label M-3”) is at 4 ° C. saved.
[0067]
(2) [Preparation of immunochromatographic strip]
An immunochromatographic strip as shown in FIGS. 1 and 2 was assembled by the following method.
[0068]
Spot 1 mg / ml of M-1, M-2, or M-3 on detection line 6 on development membrane 4 consisting of a nitrocellulose membrane (MILLIPORE, Hi-Flow Plus Membrane, HF180, 25 mm × 6 mm) did. Further, 1 μl of an anti-rabbit IgG (H + L) polyclonal antibody was spotted on the judgment line 7 and dried in an incubator at 37 ° C. for 60 minutes to immobilize the antibody. The antibody-immobilized membrane was shaken in a 1% skim milk-0.1% Triton X100 aqueous solution at room temperature for 5 minutes. Next, the membrane was taken out in a 10 mM phosphate buffer solution (pH 7.4) at room temperature for 10 minutes and then taken out, and dried in a desiccator for 60 minutes while sucking with a vacuum pump.
[0069]
Also, conjugate pad 3 (MILLIPORE, 7.5 mm × 6 mm) was taken out after shaking for 1 minute in 0.5% PVA-0.5% aqueous sucrose solution, and taken out in a desiccator for 60 minutes while sucking with a vacuum pump. Dried. A to the conjugate pad₅₂₀= 25 μl of gold colloid label M-1, gold colloid label M-2 or gold colloid label M-3 adjusted to 1.0 was added and dried in a desiccator for 60 minutes while sucking with a vacuum pump. Further, the sample pad 2 (MILLIPORE, 17 mm × 6 mm) was shaken in a 1% Tween 20-PBS aqueous solution for 1 minute and then taken out and dried in a desiccator for 60 minutes while being sucked with a vacuum pump. The absorbent pad 5 (MILLIPORE, 20 mm × 6 mm) was used untreated.
[0070]
Each component of the immunochromatography strip prepared as shown in FIG. 1 was placed on a plastic support, and an immunochromatography strip as shown in FIG. 2 was assembled. The colloidal gold label M-1 is immobilized on the nitrocellulose membrane immobilized with M-1, the colloidal gold label M-2 is immobilized on the nitrocellulose membrane immobilized with M-2, and the nitrocellulose membrane immobilized with M-3. Were combined with colloidal gold labeled M-3 to assemble an immunochromatographic strip M-1, an immunochromatographic strip M-2, and an immunochromatographic strip M-3.
[0071]
II. [Specificity evaluation test]
(1) [Saliva collection]
The subject chewed the paraffin pellet for about 1 minute and collected the secreted saliva.
[0072]
(2) [Quantification of Streptococcus mutans in saliva by culture method]
The saliva obtained according to the method of (1) above is appropriately diluted, and 100 μl is added on a solid medium and a BHI solid medium (hereinafter also referred to as “MSB”), The culture was performed at 37 ° C. under anaerobic conditions for 24-48 hours. The number of colonies generated on the MSB and BHI solid medium was counted, and the concentration of mutans streptococci was calculated from the dilution rate as cells / ml.
[0073]
The identification of Streptococcus mutans on MSB and BHI media is based on the morphological classification of colonies and, for morphologically indistinguishable colonies, serotype-specific mutans streptococci after pure culture Streptococcus mutans was identified by an immunological assay method using various antibodies and a biochemical method such as a sugar fermentation test.
[0074]
(3) [Sample pretreatment]
100 μl of saliva collected in (1) above was centrifuged and the supernatant was removed. The precipitate was suspended in 500 μl 0.1 M NaOH and the supernatant was removed by centrifugation. Next, the precipitate was suspended in 500 μl of PBS, and the supernatant was removed by centrifugation. After the same operation with PBS was performed again, 10 μl of 4M acetic acid and 5 μl of 2M sodium nitrite were added to the precipitate, mixed well, and allowed to stand at room temperature for 10 minutes. 45 μl of 1 M Tris containing 0.05% Tween 20 (pH not prepared) and 40 μl of 0.1 M phosphate buffer (pH 7.5) containing 0.05% Tween 20 were added to prepare a sample solution.
[0075]
(4) [Measurement of Streptococcus mutans by immunochromatographic strip]
I. -Using the immunochromatographic strips M-1 to M-3 produced in (2), Streptococcus mutans in the sample liquid prepared in (3) above was measured. The spot color intensity after 10 minutes on the immunochromatographic strip was identified in 4 stages (++: strong positive, ++: positive, +: weak positive,-: negative) and the culture method of (2). The number of Streptococcus mutans bacteria obtained was compared. The results are shown in Table 1.
[0076]
[Table 1]

[0077]
As is apparent from Table 1, the polyclonal antibody prepared by elution with sodium thiocyanate was 10⁵/ Ml of Streptococcus mutans could be detected, and a spot color intensity correlated with the concentration of Streptococcus mutans obtained by the culture method was obtained. From these, it can be seen that the antibodies prepared here have sufficiently low cross-reactivity with oral streptococci other than mutans streptococci.
[0078]
Example 2
[Specificity evaluation of polyclonal antibodies obtained by immunization using multiple rabbits and purification using various chaotropic ions]
(1) [Production of polyclonal antibody]
According to the method of Example 1, an antibody mixture was prepared by immunizing a cell suspension using 10 rabbits. During affinity purification with protease-treated cells in Example 1 (B) c), elution was performed with 2M sodium thiocyanate, 2M sodium iodide, or 2M sodium perchlorate, and then purified with protein A-Sepharose. did. In this way, 10 types of polyclonal antibodies were prepared for each elution method.
[0079]
(2) [Manufacture of immunochromatographic strips]
Example 1 (C) -I. According to the method described, immunochromatographic strips using the 30 polyclonal antibodies prepared in (1) above were produced.
[0080]
(3) [Specificity evaluation test]
Example 1 (C) -II. A sample liquid was prepared from saliva by the same method as described above and analyzed using the immunochromatographic strip prepared in (2) above. Table 2 shows the results of antibodies eluted with sodium thiocyanate.
[0081]
[Table 2]

[0082]
[Table 3]

[0083]
Table 3 shows the results of the antibody eluted with sodium iodide.
[0084]
[Table 4]

[0085]
[Table 5]

[0086]
The results of the antibody eluted with sodium perchlorate are shown in Table 4.
[0087]
[Table 6]

[0088]
[Table 7]

[0089]
As shown in Tables 2-4, when eluting with sodium thiocyanate, sodium iodide, sodium perchlorate,⁵/ Ml of Streptococcus mutans could be detected, and a spot color intensity correlated with the concentration of Streptococcus mutans obtained by the culture method was obtained. Irrespective of the individual rabbit, an antibody having a sufficiently low cross-reactivity with oral streptococci other than mutans streptococci was stably obtained.
[0090]
Comparative Example 1
[Specificity evaluation of polyclonal antibodies obtained by purification by acid elution using multiple rabbits]
(1) [Production of polyclonal antibody]
Affinity purification by the acid elution method was performed using each antibody mixture obtained in Example 2 (1).
[0091]
Protease-treated cells were prepared according to the method of Example 1 (A). The protease-treated cells are washed 3 times with PBS, then washed 3 times with 0.1 M glycine hydrochloride buffer (pH 2.0), and further washed 3 times with PBS.₆₀₀= 12.5) was prepared.
[0092]
Next, the antibody mixture and the protease-treated cell suspension were mixed in the same manner as in Example 1 (B) c), washed 3 times with PBS, and then 0.5 ml of 0.1 M glycine hydrochloride buffer ( The cells were suspended in pH 2.0), the adsorbed antibody was eluted, the supernatant was collected by centrifugation, and 1M Tris-hydrochloric acid (pH 9.0) was added to adjust the pH to 7.4. The same elution operation was performed 4 times, and the protein amount of each fraction was measured by absorbance at 280 nm.
[0093]
Next, the eluate was added to a column packed with 1 ml of Protein A-Sepharose (Amersham Pharmacia Biotech) previously equilibrated with PBS, and the antibody was eluted by the same method as in Example 1 (B). Polyclonal antibodies were prepared.
[0094]
(2) [Manufacture of immunochromatographic strips]
Example 1 (C) -I. According to the method described, an immunochromatographic strip using the polyclonal antibody prepared in (1) above was produced.
[0095]
(3) [Specificity evaluation test]
The antigen extract prepared in Example 3 (3) was analyzed using the immunochromatographic strip prepared in (2) above. The results are shown in Table 5.
[0096]
[Table 8]

[0097]
[Table 9]

[0098]
As shown in Table 5, it was revealed that when a polyclonal antibody was produced by affinity purification using an acid elution method, a nonspecific reaction might be detected depending on the rabbit.
[0099]
【The invention's effect】
According to the present invention, a polyclonal antibody specific for Streptococcus mutans, which is sufficiently low in cross-reactivity with oral streptococci other than mutans streptococci, is stable without being affected by individual differences in immunized animals. And can be manufactured.
[Brief description of the drawings]
FIG. 1 is a schematic view of each member of a strip used in the immunochromatography method of the present invention.
FIG. 2 is a side view of a strip used in the immunochromatography method of the present invention.
[Explanation of symbols]
1 ... Strip
2 ... Sample pad
3. Conjugate pad
4 ... Deployment membrane
5 ... Absorption pad
6 ... Detection line
7 ... Control judgment line

Claims (2)

Immunize non-human immunized animals with a) whole cells of Streptococcus mutans from which bacterial cell surface proteins have been removed by protease treatment, or b) whole cells of Streptococcus mutans to obtain an antibody mixture, A method for producing a polyclonal antibody, wherein the antibody mixture is purified by an affinity purification method,
A) When the antigen to be used for immunization to the non-human immunized animal is a), the antigen used as a carrier in the affinity purification method is a whole strain of Streptococcus mutans obtained by removing the cell surface protein by protease treatment. A body, an antigen extract of the protease-treated bacterial body, a whole cell of Streptococcus mutans, and an antigen extract of the bacterial body, and b) immunity to an immunized animal other than the human When the antigen used for b) is b), the antigen used as a carrier in the affinity purification method is the whole cell of Streptococcus mutans from which the cell surface protein has been removed by protease treatment, or the antigen extract of the protease-treated cell And
A method for producing a polyclonal antibody against Streptococcus mutans, wherein chaotropic ions are used for elution of the antibody from the carrier in the affinity purification. The method for producing a polyclonal antibody against Streptococcus mutans according to claim 1, wherein the chaotropic ion is an iodine ion, a thiocyanate ion, or a perchlorate ion.

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