JP3658857B2 - Magnetic plate for measuring agglutination reaction and its usage - Google Patents

Description

［凝集反応測定用磁石板を用いる凝集反応測定方法］
図３に前記凝集反応測定で用いるマイクロプレートの一部を断面とした側面図を示す。このマイクロプレート５２は各ウェル５４の底部がテーパー１２０度のＶ状になっており、本発明の実施例で用いるカバー板１０の座ぐり穴１０ａにウェル５４を適合保持させて使用することができる。
【００３４】
９６穴マイクロプレート５２のウェル５４内に検体と測定対象物に応じた感作磁性粒子を入れて撹拌する。
【００３５】
前記マイクロプレート５２を前記凝集反応測定用磁石板のカバー板上面に前記マイクロプレートの各ウェル５４がカバー板の座ぐり穴１０ａに対応するように安定した状態で載置させ、ウェル内の混合物が強制沈降するまで静置させる。
【００３６】
混合物を強制沈降させた後、マイクロプレート５２を前記凝集反応測定用磁石板から静かに取り外す。取り外したマイクロプレートは磁性のないところで、ウェルを傾斜させ、そのままの状態で感作磁性粒子を流れ出させるために数分置く。
【００３７】
その後、ウェル内の磁性粒子の流れ出し状態から測定を行う。
【００３８】
測定は、ウェル底部中央に感作磁性粒子が点のように収束しているときは陽性と判定し、ウェル底部から尾を引くように感作磁性粒子が流れ出していた場合には陰性と判定する。
【００３９】
［ＣＣＤカメラ方式画像測定機の作成］
（１）全体構造
図４に集磁した磁性粒子のパターンを測定するＣＣＤカメラ方式画像測定機３５の部分斜視図を示す。
【００４０】
前記ＣＣＤカメラ方式画像測定機３５は、各ウェル５４毎に撮影した画像を処理するコンピュータ３２を内蔵しており、上方に向けて設置したＣＣＤカメラ２５と、Ｘ軸方向への移動機構、カメラ上部に設置した長さ１２センチの冷陰極管を用いた光源２７、Ｙ軸方向への移動機構を持つ９６穴マイクロプレートホルダー３０とで構成されている。
【００４１】
そして、Ｘ軸方向への移動機構は、パルスモーター３７、タイミングベルト３９、及びガイドレール４０からなり、Ｙ軸方向への移動機構は、カメラと光源の間に設置したパルスモーター４２、タイミングベルト４４及びガイドレール５０からなる。
【００４２】
（２）画像データの取り込み
ＣＣＤカメラ２５を原点からＸ軸方向に移動させ、マイクロプレート５２の長辺に沿ってマイクロプレート５２の一番外側のウェル５４の画像を１列分撮影する。次に、マイクロプレート５２をＹ軸方向に前記ウェル１列分移動させ、ＣＣＤカメラ２５をＸ軸方向に移動させながら、マイクロプレート５２の２列目の各ウェルを撮影する。同様の操作をマイクロプレート５２の各列について行い、全てのウェル５４の画像データを取り込む。
【００４３】
（３）画像処理
図５に、一例として、取り込んだ各ウェル５４の画像データの各ピクセルの明暗の度合いを示す。
【００４４】
取り込んだ各ウェル５４の画像データは、５１２×１２８ピクセルのデータ量で、各ピクセルの明暗の度合いは、２５６階調で得られる（図５の（ａ）参照）。このデータを所定の明暗の閾値で２値化し、図５の（ｂ）に示すような１または０のデータに変換する。図５の（ｂ）において、１は所定の閾値より明るいデータを、０は所定の閾値より暗いデータを意味する。本実施例及び比較例につき、輝度の閾値を１５０と設定した。集磁した感作磁性粒子の直径を示す値として、２値化データの０の部分の水平方向の最長ラインを選択し、最長ライン上のピクセル数を算出した。
【００４５】
［磁石板の比較試験］
９６穴の各ウェル５４に、特公平３ー１７１０３号公報記載の磁性粒子の０．１％懸濁液を５０マイクロリットルずつ分注した前記Ｖ状の底部を有するマイクロプレート（本願出願人作製）５２を、前記磁石板１〜６上で５分間集磁し、前記ウェル５４の底部に集まった感作磁性粒子の沈降径を前記記載のＣＣＤカメラ方式画像測定装置３５にて測定した。
【００４６】
時間毎の前記磁性粒子の集合量を表２及び図６に示す。また、５分間集磁したときの磁性粒子径を１００％としたときの各集磁時間での磁性粒子集合量の割合（％）を表３及び図７に示す。
【００４７】
【表２】

【表３】

まず、表２及び図６から、前記磁石板１〜６毎に集合量がピークに達したときの集磁時間を見ると、磁石板２及び６が１．５分経過後、磁石板１が４分経過後、磁石板３、４、５は５分経過時であった。さらに、ピーク時の集合量を比較してみると、磁石板２が最も多く、６１．１ピクセルで、次に多いのが磁石板１の６０．５ピクセルであった。他の磁石３、４、５、６は５９乃至５７ピクセルであった。これらのことから磁石板２が最も短時間で最も多くの磁性粒子を集磁させることができているのが分かる。
【００４８】
また、集磁状態を見ると、磁石板２、６、５及び１においては集磁時間が０．５分経過時で磁石板３及び４より比較的多量に集磁できており、約２分経過時までは集磁量が多い。このことから、例えば、流れ出し凝集反応測定方法において、強制沈降時間を２分以内で行う場合には、磁石の磁極の向きを異ならせて挿嵌させた磁石板１、２、５及び６が、磁極の向きを同じとした磁石板３及び４より適していることが分かる。
【００４９】
また、磁石板１の構成は、支持板のみが磁石板２と異なっており、非磁性材であるアルミ板を用いている。このことから、磁性粒子の集合量及び集磁時間の双方の点で、支持板は、非磁性材のアルミ板に対して磁性材である鉄板の方が凝集反応測定において効果的であることが分かる。
【００５０】
さらに、表３及び図７と合わせて磁石板毎の効果をみると、磁石板６が、磁石板２に比べて磁性粒子の集合量は少ないものの、時間毎の集磁率では、磁石板２とほぼ同様の高い割合を示していることが分かる。ここで、磁石板４及び６は支持板において、磁石板２と同様の鉄板を用いているため、磁石板２と異なる点は、磁石８の磁極の向きのみである。このことから、磁石の磁極の向きは列毎にＳ極Ｎ極が交互になるように挿嵌させたパターンが集磁率が最も高いため、凝集反応測定用磁石板のパターンとして好ましく、磁石の磁極が１つづつ交互になるように挿嵌させたパターンにおいても、磁極を全て同じ向きに挿嵌したものと比べると、はるかに集磁率が高く凝集反応測定用磁石板として効果的であるということが分かる。
【００５１】
従って、凝集反応測定においては、隣接する磁石８の磁極の向きを異ならせて挿嵌させた磁石板とすることが強制沈降時の集磁率の面で効果的であり、かつ、支持板として、例えば鉄のような磁性材を用いた前記磁石板とすると、流れ出し凝集反応測定において、強制沈降の際の磁性粒子の集合量を従来より増加させることができる。
【００５２】
さらに、各時間毎の粒子径のバラツキを表４及び図８に示す。
【００５３】
【表４】

バラツキの点においても、磁石板２が他の磁石板と比べてバラツキの割合が低く、さらに、１分程度の短い集磁時間においてもバラツキが低いことから、集磁率および粒子径のバラツキ共に他の磁石板より優れていることが分かる。
【００５４】
また、磁石板１においても集磁時間２分経過後当たりからほぼ磁石板２と同様にバラツキが少なくなっており、上述したように集磁率も高いことから凝集反応測定に適している。
【００５５】
次に、磁石板３、４、５及び６について磁石板毎のバラツキの最も少なくなる時間を見ると、磁石板３および４は磁石板５および６に比べて約２分程度遅いことが分かる。従って、凝集反応測定を行う限られた沈降時間では、磁石板５および６が短時間で最小のバラツキで集磁することができる。ただし、磁石板４については磁石板１とほぼ同じバラツキ状態であり、これに対して同じ磁石の配設パターンの磁石板３がバラツキが最も多いことから、支持板を鉄製とした方がより効果的であることを示している。
【００５６】
以上説明したように、集磁速度の速さとウェル毎の集磁量の均一性から、磁石板１、２、５及び６が磁石板３及び４より良く、最も良い条件は磁石板２であった。
【００５７】
［流れ出し測定における磁石板の効果］
前記磁石板の比較試験で記載した磁性粒子を５０マイクロリットルずつ分注した前記マイクロプレート（本願出願人作製）５２（図３参照）と、前記磁石板２〜６を用い、前記ＣＣＤカメラ方式画像測定装置３５にて、感作磁性粒子の流れ出し状態を測定した。測定時の集磁時間は１分、傾斜時間は２分で行った。流れ出し長さ及びバラツキの結果を表５及び図９及び図１０に示す。
【００５８】
【表５】

図９において、磁石板２が流れ出しが最も長く次に磁石板４、６の順で流れ出しが長く凝集反応測定に適している。また、図１０においても磁石板６および２が同様にバラツキが少なく、磁石板４及び５においても比較的少ない。これに対して磁石板３が明らかにバラツキが目立ち、図９の粒子長も最も短かったことから凝集反応測定にはあまり適していないことが分かった。
【００５９】
［磁石板の総合評価］
これらの点から相対的に判断すると、支持板に磁性材である鉄を用い、列毎にＳ極Ｎ極が交互になるように磁石８を挿嵌した磁石板２が凝集反応測定に最も適しており、次に、同様に鉄製の支持板を用いて行及び列の隣接する磁石の磁極の向きを異ならせて挿嵌した磁石板６が適していることが分かった。
【００６０】
［応用］
上述のとおり、磁石の磁極の向きの配列パターン及び支持板の材質によって磁力の均一性及び磁力の強さを変化させることができることが明らかになった。
【００６１】
これらの変化を利用すれば、従来と同じ磁石を用いた場合でも従来より問題であった磁力の均一性を高めることができ、容器間の集磁量のバラツキを小さくすることができる。
【００６２】
また、該磁石の磁極の向きの配列と支持板の材質の組み合わせによって、１種類の磁石で何通りもの磁力を作り出すことができる。
【００６３】
このため、測定毎に性質の異なる磁性粒子を用いる場合でも、その磁性粒子に合わせて、新たに磁石を用意する必要がなく、従来の磁石の磁極の配列パターンを異ならせてその磁性粒子に適した磁力に調整して用いることができる。
【００６４】
【発明の効果】
本発明は、以上説明したように、配設した複数個の穴に隣接する磁石の磁極の向きを異ならせて該磁石を嵌挿し保持したものであるため、従来と同じ磁石を用いた場合でも磁力を均一にし、粒子径、流れ出し等のバラツキを少なくすることができると共に、磁力を強化することができ、集磁時間の短縮を図ることができる。
【００６５】
さらに、配設した複数個の穴の隣接する行又は列毎に前記磁石の磁極の向きを異ならせて挿嵌保持することで磁力をより均一かつ磁力を適度に強化することができ、効率的な測定処理が可能となる。
【００６６】
また、配設した複数個の穴の行及び列の隣接する前記磁石の磁極の向きを異ならせて挿嵌保持することでも、より磁力を均一かつ磁力を適度に強化することができ、効率的な測定処理が可能となる。
【００６７】
そして、配設された複数個の穴を有する磁石挿入板に磁石を挿嵌した磁石板本体と、該磁石板本体の一方の面に固定される支持板と、他の面に固定される前記複数個の穴に対応して座ぐり穴を設けたカバー板とからなる凝集反応測定用磁石板とすることによって、磁石を安定して保持し、容器のウェル底部に磁石を位置精度良く安定して対応させることができる。
【００６８】
そして、磁石挿入板を非磁性材とし、支持板を磁性材とすることにより、磁石間の磁力を強めると共に磁力を均一にして粒子径のバラツキを少なくすることができる。
【００６９】
また、磁石を保持する磁石挿入板をアルミ製とし、その一方の面に固定される支持板を鉄製とし、前記カバー板をアルミ製で作製したものとして磁石板を用いれば磁石の磁力を均一かつ磁力を適度に強めることができる。
【００７０】
さらに、前記カバー板において、座ぐり穴が底部に向けて次第に狭くなる傾斜穴とすれば、容器のウェルの底部と磁石との対応をより位置精度良く安定させることができる。
【００７１】
そして、凝集反応測定用磁石板を検体と、抗原又は抗体を結合した磁性粒子とを入れた容器の底部に当接して、上記磁性粒子を強制沈降させることで、磁力を均一にして、容器ウェル毎の集磁率のバラツキを少なくし、精度の良い測定を可能とできる。
【図面の簡単な説明】
【図１】 本発明の一実施例を示す凝集反応測定用磁石板１、２、５、６及び比較例の磁石板３、４の縦断面図である。
【図２】 本発明の一実施例を示す磁石板１、２、５、６及び比較例３、４の磁石板における磁石挿入時の磁極のパターンの部分平面図である。
【図３】 本発明の一実施例で用いるマイクロプレートの一部を断面とした部分側面図である。
【図４】 本発明の一実施例としての磁石板を用いたＣＣＤカメラ画像測定装置の部分斜視図である。
【図５】 ＣＣＤ画像カメラで取り込んだ各ウェルの画像データの各ピクセルの明暗の度合いを示す図である。
【図６】 磁石板１〜６の強制沈降の際の時間毎の磁性粒子の集合量を示す図である。
【図７】 磁石板１〜６において、５分間集磁したときの磁性粒子径を１００％としたときの各集磁時間での磁性粒子量の割合（％）を示す図である。
【図８】 磁石板１〜６において、各時間毎の粒子径のバラツキを示す図である。
【図９】 磁石板２〜６において、流れ出し長さ及びバラツキの結果を示す図である。
【図１０】 磁石板２〜６において、流れ出し長さ及びバラツキの結果を示す図である。
【符号の説明】
１〜６ 凝集反応測定用磁石板、 ８ 磁石、 １０ カバー板、 １０ａ 座ぐり穴、 １３ 磁石挿入板、 １３ａ 穴、 １５ 磁石板本体、 ２０，２２ 支持板、 ２５ ＣＣＤカメラ、 ２７ 冷陰極管を用いた光源、 ３０９６穴ウェルマイクロプレートホルダー、 ３２ コンピュータ、 ３５ ＣＣＤカメラ方式画像測定装置、 ３７，４２ パルスモーター、 ３９，４４ タイミングベルト、 ４０，５０ ガイドレール、 ５２ マイクロプレート、５４ ウェル。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a magnetic plate for measuring an agglutination reaction used in a measurement method (aggregation reaction measurement method) for measuring a substance to be measured using magnetic particles, and a method for using the same.
[0002]
[Prior art]
Conventionally, an immune reaction test has been performed to detect whether an antigen or antibody is present in a specimen by using an agglutination reaction caused by an antigen-antibody reaction, and in recent years, a large number of immune reactions have been performed using a magnetic plate. Methods have been developed to perform inspections simultaneously.
[0003]
These conventional magnet plates have a structure in which holes are arranged in a matrix, and magnets are held one by one in the holes, and the magnetic poles are all held in the same direction.
[0004]
As one of the agglutination measurement methods using the magnetic plate, a container having a plurality of wells corresponding to the position of the magnet on the magnetic plate is used, and a magnetic particle (antigen or antibody or antibody is bound to each of the wells) (Hereinafter referred to as sensitized magnetic particles), and a container having a plurality of wells is allowed to stand on the magnet plate, and is allowed to settle appropriately by the magnetic force of the magnet plate to cause an immune reaction from the aggregated state. A stationary method for detection was used.
[0005]
In the case of the stationary method, when an antigen or antibody is present in a specimen, a plurality of precipitated sensitized magnetic particles and the antigen or antibody bind to each other to form an aggregate. For this reason, the sensitized magnetic particles are difficult to move further and spread uniformly at the bottom of the well. On the other hand, when no antigen or antibody is present in the specimen, the sensitized magnetic particles are not bound to each other with the antigen or antibody, so no agglomerates are formed, and the sensitized magnetic particles are attracted by the magnetic force of the magnetic plate. Move and converge to the center of the bottom of the well.
[0006]
Therefore, in the determination of the agglutination reaction by the stationary method, the one that forms a distribution pattern that spreads uniformly at the bottom of the well is determined as positive, and the one that forms the distribution pattern that converges at the center of the well bottom is determined as negative. It was.
[0007]
However, in the agglutination reaction measurement method using a magnet plate, the magnetic force of the conventional magnet plate is not necessarily uniform in all parts on the plate, and in particular, the magnetic force differs between the vicinity of the magnet plate and the center of the magnet plate. As a result, the magnetic force is not uniform, and the magnetic force is not uniformly applied to each well.
[0008]
In particular, in the above stationary method, the pattern of magnetic particles formed by magnetic force is determined. Therefore, if the magnetic force is not applied uniformly to each well, the distribution pattern of the magnetic particles varies, and correct determination cannot be made. There is a risk that.
[0009]
On the other hand, in the stationary method, since the magnetism is collected over time with a weak magnetic force, there is a problem that the stationary time is long and the efficiency of processing a large number of samples is low. A new method for measuring agglutination reaction using benzene has been proposed (JP-A-3-144367, JP-A-3-191864). In this agglutination measurement method, the specimen and the sensitized magnetic particles are stirred in a container having a plurality of wells such as a microplate, and then forcedly settled by a magnetic force having a predetermined strength. This is a flow-out agglutination reaction measuring method in which the flow of magnetic particles at the bottom of the well is judged by tilting the container.
[0010]
In the flow-out agglutination reaction measurement, the sensitized magnetic particles in the specimen are forced to settle once, but if the antigen or antibody is present in the specimen, the sensitized magnetic particles and the antigen or antibody bind to each other and agglutinate. In order to form a lump, it does not move from the center of the well bottom in an agglomerated state even after tilting and hardly flows out. On the other hand, if there is no antigen or antibody in the sample, it does not bind to the sensitized magnetic particles and does not form an aggregate. Therefore, after tilting, the sensitized magnetic particles move from the bottom and flow out from the center of the bottom. .
[0011]
Therefore, the determination method in the flow-out agglutination reaction measurement determines that the magnetic particles after the well inclination form a pattern in which the magnetic particles have converged are positive, and determines that the flow pattern from the bottom center is negative. .
[0012]
In the above flow-out agglutination reaction measurement method, sensitized magnetic particles are forced to settle using a magnet having a stronger magnetic force than the magnet used in the stationary method, and the inspection time is shortened. After all, the non-uniformity of the magnetic force of the conventional magnet plate causes the strength of the agglomerated state, so that the flow after the container tilts becomes non-uniform, making the determination difficult.
[0013]
[Problems to be solved by the invention]
The present invention has been made to solve the above-described problems, and provides an agglomeration reaction measurement magnet plate and a method of using the same that can make the magnetic force of a magnet plate provided with a plurality of magnets more uniform and stronger. With the goal.
[0014]
[Means for Solving the Problems]
In order to achieve the above object, the agglomeration reaction measurement magnet plate of the present invention is one in which the magnets are inserted and held by changing the direction of the magnetic poles of the magnets adjacent to the plurality of holes arranged.
[0015]
Thereby, even when the same magnet as the conventional one is used, the magnetic force of the agglomeration reaction measurement magnet plate can be made uniform and enhanced.
[0016]
Furthermore, the agglomeration reaction measurement suitable for agglomeration reaction measurement in which the magnetic force is made uniform and strengthened by inserting and holding the magnetic poles of the magnets in different rows or columns adjacent to each other. It can be used as a magnet plate.
[0017]
In addition, for the agglutination reaction measurement suitable for the agglutination reaction measurement with the magnetic force made uniform and enhanced by inserting and holding the magnets adjacent to each other in rows and columns of the plurality of holes arranged in different directions. It can be a magnet plate.
[0018]
And the magnet plate main body which inserted the magnet in the magnet insertion plate which has a plurality of holes arranged, the support plate fixed to one surface of the magnet plate main body, and the above-mentioned fixed to the other surface A magnet plate for measuring agglomeration reaction comprising a cover plate provided with counterbore holes corresponding to a plurality of holes can be obtained.
[0019]
According to this structure, the magnet can be held more stably, and the magnet can be accurately and stably associated with the well bottom of the container.
[0020]
Then, by using the magnet insertion plate as a non-magnetic material and the support plate as a magnetic material, it is possible to increase the uniformity of the magnetic force and to increase the magnetic force between the magnets.
[0021]
If the magnet insertion plate is made of aluminum, the support plate is made of iron, and the cover plate is made of aluminum, it is possible to obtain magnet uniformity and magnetic strength suitable for agglutination reaction measurement.
[0022]
Furthermore, in the cover plate, if the counterbore hole is an inclined hole that gradually narrows toward the bottom, it corresponds to a container having a U-bottom or V-bottom well and can hold the magnet accurately and stably. .
[0023]
The magnetic plate for measuring the agglutination reaction can be used for agglomeration reaction measurement in which the magnetic particles are forced to settle by contacting the bottom of the container containing the specimen and the sensitized magnetic particles, making the magnetic force uniform, It is possible to perform measurement with higher accuracy than before by reducing variation in the magnetic flux collection ratio for each well of the container.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION AND EXAMPLES
Hereinafter, embodiments of the magnetic plate for measuring agglutination reaction according to the present invention and examples thereof will be described. Further, a case where the result of the flow-out aggregation reaction measurement method is measured by a CCD camera type image measurement device will be described based on the drawings and tables in comparison with a magnet plate in which the magnetic direction of the magnet is constant.
[0025]
[How to make a magnet plate]
FIG. 1 shows a longitudinal sectional view of agglomeration reaction measurement magnet plates 1, 2, 5, 6 and comparative examples 3 and 4 (the conditions of the magnet direction will be described later), which is an embodiment of the present invention.
[0026]
First, cover plate 10, magnet insertion plate 13, support plate 20 or support plate 22, and 96 magnets 8 are prepared.
[0027]
Here, the cover plate 10 is a plate having a thickness of 3 mm and a size of 110 mm × 74 mm, and a 96-well 54 (see FIG. 3 to be described later) of a microplate (manufactured by the present applicant) 52 is inserted thereinto. Counterbore 10a is provided correspondingly. This counterbore 10a is an inclined hole with a taper of 120 degrees that has an opening with a bottom diameter of 3 mm and gradually narrows toward the bottom. The cover plate 10 is preferably made of a nonmagnetic material such as an aluminum plate. Further, the size and thickness of the cover plate 10 and the shape of the counterbore 10a can be changed according to the microplate to be used.
[0028]
The magnet insertion plate 13 has a thickness of 1.5 mm and 110 mm × 74 mm, and is provided with a cylindrical hole 13 a having a diameter of 5 mm in accordance with the well position of 96 holes. The magnet insertion plate 13 is preferably made of a magnetic material such as iron, for example.
[0029]
The magnet 8 has a cylindrical shape with a diameter of 5 mm and a thickness of 1.5 mm in accordance with a cylindrical hole 13 a provided in the magnet insertion plate 13, and has a magnetic force of about 2000 gauss.
[0030]
The magnet plate body 15 is formed by inserting the magnet 8 into the cylindrical hole 13 a of the magnet insertion plate 13.
[0031]
Further, the support plate 20 or 22 is a plate having a thickness of 2 mm and 110 mm × 74 mm, and either a non-magnetic material or a magnetic material can be used. It can also be made of iron, or it can be made of aluminum like the support plate 22.
[0032]
Then, the magnet insertion plate 13 is stacked on the prepared support plate 20 or 22, and the magnet 8 is inserted into a cylindrical hole 13 a provided in the magnet insertion plate 13, and the magnet insertion plate 13 and the magnet 8 are formed. The magnet plate main body 15 is produced on the support plate 20 or 22, and the cover plate 10 is further stacked, and the four corners of the three plates are fixed with screws to produce an agglomeration reaction measurement magnet plate. The agglomeration reaction measurement magnet plate to be produced is produced by making the magnetic material support plate 20 or the non-magnetic material support plate 22 different in the direction of the magnetic poles of the 96 magnets 8 to produce various types of agglutination reaction magnet plates. Four types of magnet plates, ie, magnet plates 1, 2, 5, and 6, were created under the conditions shown in Table 1, and two types of magnet plates 3, 4 were created as comparative examples. FIG. 2 shows a magnetic pole pattern when the magnet plates 1 to 6 are inserted.
[0033]
[Table 1]

[Method of measuring agglutination reaction using a magnet plate for agglutination reaction measurement]
FIG. 3 is a side view showing a cross section of a part of the microplate used in the aggregation reaction measurement. The bottom of each well 54 has a V shape with a taper of 120 degrees, and the microplate 52 can be used with the well 54 fitted and held in the counterbore 10a of the cover plate 10 used in the embodiment of the present invention. .
[0034]
Sensitized magnetic particles corresponding to the specimen and the measurement object are placed in the well 54 of the 96-well microplate 52 and stirred.
[0035]
The microplate 52 is placed on the upper surface of the cover plate of the agglutination reaction measurement magnet plate in a stable state so that each well 54 of the microplate corresponds to the counterbore 10a of the cover plate. Let stand until forced settling.
[0036]
After the mixture is forced to settle, the microplate 52 is gently removed from the agglutination reaction measurement magnetic plate. The removed microplate is placed in a place where there is no magnetism, the well is tilted, and the sensitized magnetic particles are allowed to flow for a few minutes in the same state.
[0037]
Thereafter, measurement is performed from the flow-out state of the magnetic particles in the well.
[0038]
In the measurement, when the sensitized magnetic particles converge at the center of the well bottom like a dot, it is determined to be positive, and when the sensitized magnetic particles have flowed out from the bottom of the well, it is determined to be negative. .
[0039]
[Creation of CCD camera type image measuring machine]
(1) Overall Structure FIG. 4 shows a partial perspective view of a CCD camera type image measuring machine 35 that measures the pattern of magnetic particles collected.
[0040]
The CCD camera type image measuring machine 35 has a built-in computer 32 for processing an image taken for each well 54, a CCD camera 25 installed upward, a moving mechanism in the X-axis direction, and an upper part of the camera. And a 96-well microplate holder 30 having a moving mechanism in the Y-axis direction, using a 12-cm-long cold cathode tube installed in the Y-axis.
[0041]
The movement mechanism in the X-axis direction includes a pulse motor 37, a timing belt 39, and a guide rail 40. The movement mechanism in the Y-axis direction includes a pulse motor 42 and a timing belt 44 installed between the camera and the light source. And a guide rail 50.
[0042]
(2) Image Data Acquisition The CCD camera 25 is moved from the origin in the X-axis direction, and an image of the outermost well 54 of the microplate 52 is taken for one column along the long side of the microplate 52. Next, the microplate 52 is moved by one row of the wells in the Y-axis direction, and each well in the second row of the microplate 52 is photographed while the CCD camera 25 is moved in the X-axis direction. The same operation is performed for each row of the microplate 52, and the image data of all the wells 54 are captured.
[0043]
(3) Image Processing FIG. 5 shows, as an example, the degree of brightness of each pixel of the captured image data of each well 54.
[0044]
The captured image data of each well 54 is obtained with a data amount of 512 × 128 pixels, and the degree of brightness of each pixel is obtained with 256 gradations (see FIG. 5A). This data is binarized with a predetermined brightness / darkness threshold value and converted into 1 or 0 data as shown in FIG. In FIG. 5B, 1 means data brighter than the predetermined threshold, and 0 means data darker than the predetermined threshold. For this example and the comparative example, the luminance threshold was set to 150. As the value indicating the diameter of the collected sensitized magnetic particles, the longest horizontal line of the 0 portion of the binarized data was selected, and the number of pixels on the longest line was calculated.
[0045]
[Magnetic plate comparison test]
A microplate having the V-shaped bottom portion prepared by dispensing 50 microliters of a 0.1% suspension of magnetic particles described in Japanese Patent Publication No. 3-17103 into each well 54 of 96 holes (manufactured by the present applicant) 52 was collected on the magnetic plates 1 to 6 for 5 minutes, and the sedimentation diameter of the sensitized magnetic particles collected on the bottom of the well 54 was measured by the CCD camera type image measuring device 35 described above.
[0046]
The amount of the magnetic particles aggregated over time is shown in Table 2 and FIG. Further, Table 3 and FIG. 7 show the ratio (%) of the amount of magnetic particles aggregated at each magnetic collection time when the magnetic particle diameter when collecting the magnetic flux for 5 minutes is 100%.
[0047]
[Table 2]

[Table 3]

First, from Table 2 and FIG. 6, when looking at the magnetism collection time when the aggregate amount reaches the peak for each of the magnet plates 1 to 6, after 1.5 minutes have elapsed for the magnet plates 2 and 6, the magnet plate 1 is After the elapse of 4 minutes, the magnetic plates 3, 4, 5 were at the time of 5 minutes. Further, comparing the amount of gathering at the peak, the magnet plate 2 was the most, 61.1 pixels, and the next most was 60.5 pixels of the magnet plate 1. The other magnets 3, 4, 5, 6 were 59 to 57 pixels. From these facts, it can be seen that the magnet plate 2 can collect the most magnetic particles in the shortest time.
[0048]
Further, when looking at the magnetism collecting state, the magnet plates 2, 6, 5 and 1 were able to collect a relatively large amount of magnets than the magnet plates 3 and 4 when the magnetizing time was 0.5 minutes, and about 2 minutes. A large amount of magnetism is collected until the time has elapsed. From this, for example, in the flow-out aggregation reaction measurement method, when the forced sedimentation time is performed within 2 minutes, the magnet plates 1, 2, 5, and 6 inserted with the magnetic poles of different orientations inserted, It can be seen that the magnetic plates 3 and 4 having the same magnetic pole direction are more suitable.
[0049]
The configuration of the magnet plate 1 is different from the magnet plate 2 only in the support plate, and uses an aluminum plate that is a nonmagnetic material. Therefore, in terms of both the aggregate amount of magnetic particles and the magnetic collection time, the support plate is more effective in the agglutination reaction measurement than the non-magnetic aluminum plate as the magnetic plate. I understand.
[0050]
Furthermore, when the effect for every magnet plate is seen together with Table 3 and FIG. 7, although the magnet plate 6 has a smaller amount of magnetic particles than the magnet plate 2, the magnet plate 2 and It can be seen that the ratio is almost the same. Here, since the magnet plates 4 and 6 use the same iron plate as the magnet plate 2 in the support plate, the only difference from the magnet plate 2 is the direction of the magnetic pole of the magnet 8. From this, the direction of the magnetic pole of the magnet is preferably the pattern of the agglomeration reaction measurement magnet plate because the pattern in which the S pole and the N pole are alternately inserted for each row has the highest magnetic flux collection. Even in a pattern in which the magnetic poles are alternately inserted one by one, the magnetic flux collection is much higher than that in which all the magnetic poles are inserted in the same direction, and it is effective as a magnet plate for measuring an agglutination reaction. I understand.
[0051]
Therefore, in the agglomeration reaction measurement, it is effective in terms of magnetic flux collection at the time of forced settling to make a magnet plate inserted with the magnetic poles of the adjacent magnets 8 different in direction, and as a support plate, For example, when the magnetic plate using a magnetic material such as iron is used, the aggregate amount of magnetic particles during forced sedimentation can be increased in the flow-out aggregation reaction measurement.
[0052]
Furthermore, the dispersion of the particle diameter for each time is shown in Table 4 and FIG.
[0053]
[Table 4]

In terms of variation, the magnet plate 2 has a low variation rate compared to other magnet plates, and furthermore, variation is low even in a short magnetizing time of about 1 minute. It turns out that it is superior to the magnet plate.
[0054]
Further, the magnet plate 1 also has little variation after the magnetizing time of 2 minutes has passed, as in the case of the magnet plate 2, and is also suitable for the agglutination reaction measurement because of the high magnetic flux collecting rate as described above.
[0055]
Next, when the time when the variation of each magnet plate is minimized for the magnet plates 3, 4, 5, and 6 is seen, the magnet plates 3 and 4 are about 2 minutes slower than the magnet plates 5 and 6. Accordingly, the magnet plates 5 and 6 can collect magnetism with minimum variation in a short time with a limited settling time during which the aggregation reaction measurement is performed. However, the magnet plate 4 is almost the same in variation state as the magnet plate 1, and the magnet plate 3 having the same magnet arrangement pattern has the largest variation. On the other hand, the support plate made of iron is more effective. It shows that
[0056]
As described above, the magnet plates 1, 2, 5 and 6 are better than the magnet plates 3 and 4 from the speed of the magnetism collection speed and the uniformity of the amount of magnetism in each well, and the best condition is the magnet plate 2. It was.
[0057]
[Effect of magnetic plate on flow-out measurement]
Using the microplate (manufactured by the present applicant) 52 (see FIG. 3) into which 50 microliters of magnetic particles described in the comparative test of the magnetic plate were dispensed and the magnetic plates 2 to 6, the CCD camera system image With the measuring device 35, the flow-out state of the sensitized magnetic particles was measured. The magnetic collection time at the time of measurement was 1 minute, and the inclination time was 2 minutes. The results of the flow-out length and variation are shown in Table 5, FIG. 9 and FIG.
[0058]
[Table 5]

In FIG. 9, the magnet plate 2 has the longest flow out and then the magnet plates 4 and 6 have the long flow out and is suitable for the agglutination reaction measurement. Also in FIG. 10, the magnet plates 6 and 2 have the same variation, and the magnet plates 4 and 5 have relatively few variations. On the other hand, the magnetic plate 3 was clearly not uniform, and the particle length in FIG. 9 was the shortest. Therefore, it was found that the magnet plate 3 was not very suitable for the aggregation reaction measurement.
[0059]
[Comprehensive evaluation of magnet plate]
Judging relatively from these points, the magnet plate 2 in which the magnetic material iron is used for the support plate and the magnets 8 are inserted so that the S poles and the N poles are alternated for each row is most suitable for the agglutination reaction measurement. Next, it has been found that a magnet plate 6 that is similarly inserted using a support plate made of iron so that the magnetic poles of adjacent magnets in rows and columns are different in orientation is suitable.
[0060]
[application]
As described above, it has been clarified that the uniformity of the magnetic force and the strength of the magnetic force can be changed by the arrangement pattern of the magnetic pole directions of the magnet and the material of the support plate.
[0061]
If these changes are used, even when the same magnet as the conventional one is used, the uniformity of the magnetic force, which has been a problem, can be increased, and the variation in the amount of magnetic collection between the containers can be reduced.
[0062]
In addition, a number of different magnetic forces can be created with one type of magnet by combining the orientation of the magnetic poles of the magnet and the material of the support plate.
[0063]
For this reason, even when magnetic particles with different properties are used for each measurement, it is not necessary to prepare a new magnet according to the magnetic particles, and suitable for the magnetic particles by changing the magnetic pole arrangement pattern of the conventional magnet. The magnetic force can be adjusted and used.
[0064]
【The invention's effect】
As described above, the present invention is such that the magnets are inserted and held by changing the direction of the magnetic poles of the magnets adjacent to the plurality of holes arranged, so that even when the same magnet is used as before, It is possible to make the magnetic force uniform and to reduce variations in particle diameter, flow-out, etc., to enhance the magnetic force, and to shorten the time for collecting magnets.
[0065]
Furthermore, the magnetic force can be more uniformly and moderately strengthened by inserting and holding the magnetic poles of the magnets in different rows or columns adjacent to each other. Measurement processing becomes possible.
[0066]
In addition, even by inserting and holding the magnetic poles of the magnets adjacent to each other in rows and columns of a plurality of arranged holes, the magnetic force can be more uniform and moderately strengthened. Measurement processing becomes possible.
[0067]
And the magnet plate main body which inserted the magnet in the magnet insertion plate which has a plurality of holes arranged, the support plate fixed to one side of the magnet plate main body, and the above-mentioned fixed to the other side By using a magnet plate for agglomeration reaction measurement consisting of a cover plate with counterbore holes corresponding to a plurality of holes, the magnet can be stably held and the magnet can be stably positioned at the bottom of the well of the container. Can respond.
[0068]
Further, by using the magnet insertion plate as a non-magnetic material and the support plate as a magnetic material, the magnetic force between the magnets can be increased and the magnetic force can be made uniform to reduce the variation in particle diameter.
[0069]
Further, if the magnet insertion plate for holding the magnet is made of aluminum, the support plate fixed to one surface thereof is made of iron, and the cover plate is made of aluminum, and the magnet plate is used, the magnetic force of the magnet is uniform and Magnetic force can be increased moderately.
[0070]
Further, in the cover plate, if the counterbore is an inclined hole that gradually narrows toward the bottom, the correspondence between the bottom of the well of the container and the magnet can be stabilized with higher positional accuracy.
[0071]
Then, the magnetic plate for agglutination reaction measurement is brought into contact with the bottom of the container containing the specimen and the magnetic particles to which the antigen or antibody is bound, and the magnetic particles are forcibly settled, so that the magnetic force becomes uniform and the container well It is possible to reduce the variation in the magnetic flux collection rate and to perform accurate measurement.
[Brief description of the drawings]
FIG. 1 is a longitudinal sectional view of agglomeration reaction measuring magnet plates 1, 2, 5, 6 and a comparative magnet plate 3, 4 showing an embodiment of the present invention.
FIG. 2 is a partial plan view of a magnetic pole pattern at the time of magnet insertion in the magnet plates 1, 2, 5, 6 and Comparative Examples 3, 4 showing an embodiment of the present invention.
FIG. 3 is a partial side view showing a cross section of a part of a microplate used in an embodiment of the present invention.
FIG. 4 is a partial perspective view of a CCD camera image measurement device using a magnet plate as one embodiment of the present invention.
FIG. 5 is a diagram showing the degree of lightness and darkness of each pixel of image data of each well captured by a CCD image camera.
FIG. 6 is a diagram showing the aggregate amount of magnetic particles per time when the magnetic plates 1 to 6 are forced to settle.
FIG. 7 is a graph showing the ratio (%) of the amount of magnetic particles at each magnetic collection time when the magnetic particle diameter when magnetizing for 5 minutes in the magnet plates 1 to 6 is taken as 100%.
FIG. 8 is a diagram showing a variation in particle diameter for each time in the magnet plates 1 to 6;
FIG. 9 is a diagram showing the results of flow-out length and variation in the magnet plates 2 to 6;
FIG. 10 is a diagram showing the results of flow-out length and variation in the magnet plates 2 to 6;
[Explanation of symbols]
1-6 Magnet plate for agglutination reaction measurement, 8 magnet, 10 cover plate, 10a counterbore, 13 magnet insertion plate, 13a hole, 15 magnet plate main body, 20, 22 support plate, 25 CCD camera, 27 cold cathode tube Light source used, 3096 well microplate holder, 32 computer, 35 CCD camera type image measuring device, 37, 42 pulse motor, 39, 44 timing belt, 40, 50 guide rail, 52 microplate, 54 well.

Claims (6)

A magnet plate for measuring agglutination reaction,
A magnet plate main body in which the magnet is inserted by changing the direction of the magnetic pole of the magnet adjacent to the magnet insertion plate having a plurality of holes disposed ;
A support plate fixed to one surface of the magnet plate body;
A cover plate provided with counterbore holes corresponding to the plurality of holes fixed to the other surface,
A magnet plate for measuring agglutination reaction, wherein the magnet insertion plate is made of a non-magnetic material and the support plate is made of a magnetic material .   2. The magnetic plate for measuring agglutination reaction according to claim 1, wherein the magnets are inserted and held by changing the direction of the magnetic pole of each of the adjacent rows or columns of the plurality of holes.   The agglomeration reaction measurement magnet plate according to claim 1, wherein the magnetic poles of the magnets adjacent to each other in rows and columns of the plurality of holes are inserted and held in different directions. The magnet plate for agglutination reaction measurement according to any one of claims 1 to 3, wherein the magnet insertion plate is made of aluminum, the support plate is made of iron, and the cover plate is made of aluminum. In the cover plate, agglutination reaction measurement magnet plate according to any one of claims 1 to 4 counterbore is gradually narrower inclined hole towards the bottom. The aggregation reaction measuring magnet plate abuts against the bottom of the container containing a sample and a sensitized magnetic particles, for agglutination reaction measurement according to any one of the claims 1 to 5 the magnetic particles are forced sedimentation How to use the magnet plate.

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