JP2005043049A - Method for detecting or determining quantitatively biological reaction using superparamagnetic labeling - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect the generation of a biological reaction using a superparamagnetic particle, or to determine result thereof quantitatively. <P>SOLUTION: The same biomolecule of a member of a biological coupling pair is coupled or adsorbed onto the superparamagnetic particle to contact with a liquid or solid sample wherein the molecule of a biological coupling partner is known to be contained in the biomolecule, or is estimated therein. The coupling object or the adsorption object is brought into contact with the liquid or solid sample for a sufficient time to form a firmly coupled three-dimensional block containing the connected biomolecule and the coupled superparamagnetic particle. The block is exposed to a magnetic field of an experiment equipment for the shortest time required for inducing magnetization in the superparamagnetic particle, and the magnetic field is removed immediately. The block exhibits the nonpermanent measurable magnetization lasting for at least 20 minutes after exposed to the magnetic field. In this method, the bioreaction is detected using a correlation between magnetic signal intensity and one quantitative determination concentration or the number of the biomolecule. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

「磁気記録」は、電磁単位（ｅｍｕ）である。
【００８６】
別のシリーズの実験は、最初の記録を３．５分に行ったこと以外は、全く同じに行った、３．５分とはＳＱＵＩＤの磁界が１テスラから０に復帰するまでに必要な時間であり、この後に４．０分で２回目の記録を行い、４．５分で３回目の記録を行った。結果を表２に示し、空欄は、記録を残さなかったことを示している。
【表２】

【００８７】
表３は、当初の溶液における抗原レベルがそれぞれ、５０ ｎｇ／ｍｌ、１００ ｎｇ／ｍｌおよび２００ ｇ／ｍｌであった別のシリーズの実験の結果を示す。他の全ての局面において、このシリーズの実験は、その結果を表２に示す実験と全く同じに行った。
【表３】

【００８８】
さらなるシリーズの実験は、（１）溶液の抗原レベルがそれぞれ、１ｎｇ／ｍｌ、１０ ｎｇ／ｍｌ、および２００ ｎｇ／ｍｌであったこと、および（２）乾燥した部分ではなくて湿った部分について記録を行ったこと以外は、表２および３において記録を得た実験と同じように行った。結果を表４に示す。
【表４】

【００８９】
実施例５
現在利用可能な多くの機器は一定の磁界に限って操作し、磁界は存在する超常磁性粒子を磁化するために最初に適用され、測定のあいだも残っているに違いないことから、試験全体を通してＳＱＵＩＤを１テスラの一定の磁界で維持して、実施例４の表１、２、３、および４のそれぞれに示した実験と同じように平行した実験を行うことを決定した。以下の表１Ａは、表１における対応する抗原濃度の試料と同一に調製した試料に関して１テスラの一定磁界下で得られた記録を示す。
これらの試料は、表１にその記録を示す試料と同様に、それぞれの試料を磁界に最初１０秒間曝露後、５．０分で測定した：
【表１Ａ】

【００９０】
表２Ａは、一定の磁界が全体を通して維持される、表２の試料と同一に調製した試料について得られた記録を示す。この表において、記録＃１は、それぞれの試料を一定の磁界に最初１０秒間暴露後３．５分に得たが、＃２の記録は、４．０分で得て、記録＃３は４．５分で得た。
【表２Ａ】

【００９１】
表３Ａは、表３の試料と同一に調製された試料について得られた記録を示し、記録は表２Ａに明記した同じ磁界および間隔で得た。
【表３Ａ】

【００９２】
表４Ａは、表４の記録を得た対応する試料と全く同じように調製した湿潤部分について同じ磁界下で得た記録を示す。
【表４Ａ】

【００９３】
実施例４に示す様々なデータにおいて、それぞれがＩＣＴ片の捕獲域を含む湿潤部分について得た表４の記録は、非永続的集合磁化が長期間にわたってより大きい程度の強度に保持され、少なくともＳＱＵＩＤ機器によって測定を行った場合、対応する乾燥試料より、これらの「湿潤部分」または「湿潤片」試料ではより顕著な用量依存性を示す。これは、図７および８を比較することによってより容易に認められ、図７では、表１における乾燥片の記録＃１と＃２に関して、抗原濃度に対するＭ、すなわちの電磁単位、のデータをグラフに示し、表４の湿潤片記録＃１に関して、データを同様にグラフに示す。
【００９４】
意外にも、一定磁界でＳＱＵＩＤにおいて記録を得た実施例５のデータも同様に、対応する乾燥試料より「湿潤部分」または「湿潤片」試料に関してはるかに顕著な用量依存的反応を示す。この場合も、表４Ａからの湿潤片記録を同様にグラフに示した図１０と比較して、表１Ａの乾燥片の記録＃１および＃２に関して、抗原濃度に対する先に定義したＭをグラフに示した図９において、容易に認められうる。
【００９５】
これらの知見は、調べる試料を湿潤状態で維持すると、特にノイズに対するシグナルの比を最小限にして、したがって感度を最大限にすることに関して、優れた性能を得ることができることを示している。この現象は磁化後に測定した非永続的集合磁化および磁界の除去後の測定のみならず、測定のあいだ一定の磁界を用いるといった、より一般的な測定系にも関係する。
【００９６】
本発明は、イヌの疾患であるイヌ糸状虫（Ｄｉｒｏｆｉｌａｒｉａ ｉｍｍｉｔｉｓ）の原因物質の抗原に対して特異的な周知の免疫診断系の状況において例示してきたが、本発明により広い範囲の応用がもたらされ、測定することが難しいもしくは不可能であるとこれまで思われていた観察現象の結果が大きく改善され、または正確な定量的測定が可能となることは、免疫化学および／または生物学の当業者に容易に明らかとなると思われる。したがって、本発明の範囲は、添付の特許請求の範囲によってのみ制限されると解釈される。
【００９７】
【発明の効果】
本発明により、超常磁性粒子を用いて生物学的反応の発生を検出する、またはその結果を定量する方法が提供された。
【図面の簡単な説明】
添付の図面は以下の通りである：
【図１】図１は、文献から得た典型的な強磁性材料に関するヒステリシス曲線の略図である。略図において、Ｈは適用した磁界をエルステッドで表し、σはそこから得られた磁化を表す。磁化の飽和は、σｓによって表し、磁界を除去後に残っている残留または磁化をσｒで表す。σｒをゼロにするために必要な逆磁界は保磁力Ｈｃによって表す。
【図２】図２は、小さいポリアクリル酸によってコーティングした後、ベチルと呼ばれる市販の抗体に反応させた超常磁性粒子について測定したヒステリシスを重ね合わせたプロットである。そのような三つの測定は、粒子の浮遊液について測定した異なる３つの鉄濃度、すなわち２．４ ｍｇ／ｍｌ（プロット上の●）、１．０ ｍｇ／ｍｌ（プロット上の■）、および０．３ ｍｇ／ｍｌ（プロット上の▲）の同一に処置した粒子上で行った。プロットにおいて、Ｍはｅｍｕ（電磁単位）／ｃｃ（「ｃｍ^３」）で表した総磁化を表し、Ｈは適用した磁界をエルステッドで表し、Ｈｃは磁化を負荷するため（ゼロの線から右方向）、または磁化を逆転させるため（ゼロの線から左方向）に必要な保磁力をエルステッドで表したものである。図２Ａは、ポリマーをコーティングした（すなわち小さいポリアクリル酸コーティングまたはコンドロイチン硫酸Ａコーティング）超常磁性Ｆｅ_３Ｏ_４粒子上で２つの異なる機器測定技術を用いて、重なり合わせたヒステリシス測定の同様のプロットであり、コーティング前の粒子は本明細書に記載の実験的研究において用いたものと同一であり、ヒステリシス測定は振動試料磁力計（「ＶＳＭ」）によって、ＣＰＳ抗体に結合したＦｅ_３Ｏ_４の超常磁性粒子について行った。図２Ａにおいて、ＳＱＵＩＤ機器を用いて行ったポリマーコーティングした超常磁性Ｆｅ_３Ｏ_４粒子の測定は、■によって表し；ポリマーをコーティングした超常磁性Ｆｅ_３Ｏ_４粒子について行った測定は、○で表し、および超常磁性Ｆｅ_３Ｏ_４粒子−抗体結合物の測定は、▲によって表す。
【図３】図３は、ＩＣＴ装置の捕獲ラインで測定した２つのシリーズの超常磁性粒子−抗原−固定抗体サンドイッチの、抗原濃度ｎｇ／ｍｌに対する相対的磁気単位について、測定した磁化をプロットした図である。このタイプのＩＣＴ装置は、ハワード・チャンドラー（Ｈｏｗａｒｄ Ｃｈａｎｄｌｅｒ）の米国特許出願番号第０７／７０６，６３９号、現在では米国特許第６，１６８，９５６号、またはその様々な係属特許および出願の如何なるものにも記載され、その全てがスミスクラインディアクノスティック社（Ｓｎｉｔｈ Ｋｌｉｎｅ Ｄｉａｇｎｏｓｔｉｃｓ， Ｉｎｃ）に譲渡されるが、本発明の代理人であるビナックスインクに対し、広い物質領域に関する免疫アッセイ法のために独占的に認可された。これらのデータは実施例３に記載の研究から得た。
【図４】図４は、実施例３において用いた超常磁性粒子標識のＦｅ_３Ｏ_４としてμｇ／ｍｌで計算されたＦｅ濃度を、測定したＣＨＷ抗原濃度ｎｇ／ｍｌに対してプロットした図である。このデータは、実施例３に記載した研究から得た。
【図５】図５は、抗原濃度ｐｇ／ｍｌに対する相対的磁気単位について、測定した磁化をプロットした図である。同様に、実施例３に記載した研究からのデータを体現している。
【図６】図６は、本明細書に記述した実験的研究において用いた超常磁性Ｆｅ_３Ｏ_４粒子（パターンＡ）および上記の２つのポリマーの一つによりコーティングした後の超常磁性Ｆｅ_３Ｏ_４粒子（パターンＢ）の粒子サイズを測定して重なり合わせた結果を示すＸ線回折略図である。
【図７】図７は、実施例４の表２の記録１および２（試料シリーズ１および２）からの乾燥片データについて、抗原濃度に対し電磁単位でＭをプロットした図である。
【図８】図８は、実施例４の表４の記録１シリーズからの湿潤片データを用いて、抗原濃度に対し同じ単位でＭをプロットした図である。
【図９】図９は、実施例５の表２Ａの試料の記録１および２シリーズの乾燥片データから、図７と同様にプロットした図である。
【図１０】図１０は、実施例５の表４Ａの試料の記録１シリーズの湿潤片データから、図８と同様にプロットした図である。[0001]
This application was filed on Oct. 20, 2001, which is a continuation-in-part of U.S. Patent Application No. 09 / 692,463, filed October 20, 2000, but now abandoned. This is a continuation-in-part of pending US patent application Ser. No. 09 / 978,105.
[0002]
BACKGROUND OF THE INVENTION
The present invention relates to novel detection systems that recognize and / or monitor biological reactions, including biochemistry, and in particular immunochemical reactions. Introducing superparamagnetic forces into this system effectively facilitates the accurate detection of the extent of such a reaction based on the concentration or number of molecules of a single reactant involved in the reaction. The invention covers other areas where biological reactions are currently observed or measured, including immunoassays, chromatographic separation of molecules, nucleic acid probe analysis, pesticide residue analysis, oligonucleotide probes, and biochemistry, or still It relates to a variety of in vitro applications, including areas not reported but where such observations and measurements can be usefully performed.
[0003]
[Prior art]
According to various references, certain colloidal metal particles, and especially gold colloids, can be used as identifiers for unusual cellular or molecular entities that may be present in biological tissues, and for various immunoassays, nucleic acid probe analysis, chromatography Separation by oligonucleotide, monitoring of biological reactions, identification of organisms causing one or more diseases, possible participation in a specific biological reaction or disrupting the normal function of such a reaction Tags for biomolecules containing biochemistry in the identification of specific specific entities, identification of entities that elicit pathological responses, and myriad other specific applications for similar information on bio / biochemical properties It is described to be used as.
[0004]
The use of colloidal gold to tag these reactions may be done with care and with the addition of complex conditions and steps to provide somewhat quantitative property information. However, these colloidal gold tags are more easily used than qualitative attempts to obtain quantitative information with them, and generally produce more accurate and useful results. In another aspect, colloidal gold exhibits significant advantages as a tag material, especially when the goal is to use it to identify specific biological molecules or entities, but only colloidal gold tags Obtaining reliable quantitative information from the assays used and other reactions is often a very difficult and time consuming task.
[0005]
There are many suggestions regarding the use of magnetic beads or particles as labels for biological reactions, assuming that the magnetic field sensor produces a record that can determine, for example, the size of the molecule or the yield of the desired end product. Has been made. See, for example, Adelmann, US Pat.
[0006]
Adelman magazine article states that off-the-shelf magnetic beads are “residual [sic] magnetized, ie permanently magnetizable (page 33), and their residual magnetic field quantifies polynucleotides and other bound targets. In that example, what is called a bead is one that is unusually large in size, on the order of 800 nm, and some that is 4 microns in size. Such large beads, because of their rheological properties, are not easily moved through the normal somewhat viscous medium where such reactions occur, and also because such reactions are Often they do not flow easily along a matrix such as cellulose derivatives, paper, wood, glass etc. In the paper, scientists from biomedical and biotechnology laboratories bound to target whole cells, DNA, or proteins when using magnetic beads. It has long been recognized that there is a need to separate excess unbound beads from beads, and this separation is performed by subjecting the mixture to a fixed magnetic field that attracts unbound beads (Id. Both the permanent magnetization of these beads and the situation where a fixed magnetic field attracts an excessive amount of beads strongly suggests that the bead features are ferromagnetic and not superparamagnetic. is doing.
[0007]
US Pat. No. 6,057,086, for example, proposes to use a magnetoresistive element similar to that used to read magnetic tapes or disks in electronic and computer applications, for example, It is described that about 20 × 20 μm (6th paragraph, 34th line) is measured in order to detect a large number of particles per element (also distinguished from the proposed one-element-one-element mode). This magnetoresistive element is first pre-coated with an insulator to covalently bind a binding molecule specific for the target molecule. The element prepared in this way may be ferromagnetic, ferrimagnetic, or superparamagnetic after being placed in a flow cell to which a liquid sample containing the target molecule is added, but specific binding to the target molecule. Add a suspension of particles 1-5 nm in diameter with a molecular coating. The specification describes that each magnetoresistive element has the purpose of counting particles that bind to the target molecule, and before activating the magnetoresistive sensing element, using a magnetic device such as an electromagnet It is disclosed to remove particles that adhere to each other (7th paragraph, lines 1 to 14). This states that this function is best provided by sending a short (-10-100 ms) pulsed current created by a capacitive discharge circuit through an air-core electromagnetic coil (Id.). 11th to 14th lines). Next, a magnetic field generator (7th paragraph, 21st to 38th lines) magnetizes the bound beads, each of which generates a magnetic field, changes the resistance of the magnetoresistive element that binds to it, and then the resistance Compare the control element with the Wheatstone bridge, digitize the data and send it to the microprocessor, where the total number of beads on a particular magnetoresistive element is determined and the target molecule concentration is calculated therefrom Can do.
[0008]
Various other methods using magnetic or paramagnetic beads to measure the target substance in the sample have been described as well. See, for example, Rapoport, US Pat. No. 6,057,031, which is characterized as a paramagnetic, diamagnetic, or ferromagnetic labeling material (“paramagnetic”) that binds at least partially to a target substance. Measuring the change in magnetic susceptibility of a liquid sample using a conductor when the latter, which represents oxygen, which may be present, is subjected to an applied magnetic field.
[0009]
The German group Kotits has shown that ferromagnetic (including ferrimagnetic) nanoparticles can be used to tag various biomolecules. See Kotitz et al., Non-Patent Document 2, at the 41st Annual Conference on Magnetic Materials and Magnetic Materials held in Atlanta, Georgia in November 1996. It relates to published academic papers and related patent document 4, claiming the German priority date of January 27, 1995 and naming the same four inventors as the abstract authors. Similarly, further academic papers from the same group published in Non-Patent Document 3 are closely related to these published abstracts and US patents, the first paper was August 1996, Pittsburgh, Pennsylvania. Was presented at the 1996 Conference on Applied Superconductivity, and the group later published a paper as Non-Patent Document 4. In all four of these references, the group studied ferromagnetic nanoparticles containing ferrimagnetism rather than superparamagnetic particles. Three of these articles, including US patents, relate to the detection of analytes labeled with these ferromagnetic materials using a detection method that the authors (inventors) call “magnetic relaxation measurements”. SQUIDS measures the time in milliseconds after ferromagnetic particles are subjected to at least partial rearrangement of their magnetic moments, both after exposure to a magnetic field, usually done in a magnetically shielded environment, and after removal of the magnetic field. Used for.
[0010]
The IEEE bulletin paper relates to using a SQUID to measure the remanent magnetization of a sample in the absence of an external magnetic field. More specifically, this paper describes a method for coupling monoclonal antibodies against type III collagen to dextran-coated ferromagnetic iron oxide nanoparticles with an average diameter of 13 nm. At the same time, the polystyrene tube was incubated with a PBS solution of type III collagen, thereby adsorbing this antigenic material to the wall of the tube.
[0011]
Ferromagnetic nanoparticle labeled monoclonal antibody in ferrofluid was added to these prepared tubes and incubated for 60 minutes. Each tube was exposed to a magnetic field for 10 seconds. Next, the residual magnetism was measured in a tube filled with ferrofluid, and the measurement was performed in a magnetic shielding environment. The tubes were then decanted and washed 3 times with PBS, and the remanence was measured again for each. The results showed no change in residual signal compared to the results obtained while the tube was filled with ferrofluid. From this, all unbound particles, ie particles not involved in the antibody-antigen reaction, can be ignored regardless of whether they first react with the monoclonal antibody alone to form “blocked particles”. It was concluded that the residual effect that was possible and was only observed with the particle-targeted antibody bound to the antigen adsorbed on the cell wall. Moreover, this measured remanence was found to be a linear function of antigen concentration on the tube wall.
[0012]
In the present invention, individual superparamagnetic particles of physical size exhibiting an average diameter of 1 to about 100 nm, preferably 1 to about 60 nm, and most preferably 5 nm to 50 nm are not permanently magnetizable. (Thus, it does not have the remanence described in the IEEE literature) but it is nevertheless long enough to make a very useful measurement with a large amount of information when closely packed into a connected bio-organic matrix. Includes the discovery of obtaining a lasting non-permanent magnetization effect.
[0013]
[Patent Document 1]
US Pat. No. 5,656,429
[Patent Document 2]
US Pat. No. 5,981,297
[Patent Document 3]
US Pat. No. 5,978,694
[Patent Document 4]
US Pat. No. 6,027,946
[Non-Patent Document 1]
Adelman, L.M. "J. Assn. For Laboratory Automation 4", June 1999, No. 3, pp. 32-35.
[Non-Patent Document 2]
Kotitz et al., Abstract: Magnetic Conductivity Measurement of Magnetic Nanoparticles Based on Superconducting Quantum Interference Devices as a Novel Tool for Binding-Specific Detection of Biological Binding Reactions Tool for the Binding Specific Detection of Biological Binding Reactions), “J. Appl. Phys.”, April 1997, 81, 4317.
[Non-Patent Document 3]
Kotitz et al., "IEEE Transaction on Applied Superconductivity", 1997, Vol. 7, No. 2, 3rd edition, pages 3678-3681
[Non-Patent Document 4]
Kotitz et al., “J. Magnetics and Magnetic Materials”, April 1999, Vol. 194, Nos. 1-3, 62-68.
[0014]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for detecting the occurrence of a biological reaction or quantifying the result using superparamagnetic particles.
[0015]
[Means for Solving the Problems]
The present invention relates to physical properties measured by X-ray diffraction and transmission electron microscopy, wherein the average diameter ranges from 1 to about 100 nm, preferably from 1 to about 60 nm, and most preferably from about 5 nm to 50 nm. Using a superparamagnetic particle having a size as a labeling substance for at least one selected or suspected reactant of a biological reaction, wherein the reactant is capable of interacting within or between reactants. When it occurs, it forms a three-dimensional mass of closely packed, often molecularly cross-linked bio-organic materials and associated superparamagnetic (SPM) particles. In such a mass, the SPM particles are in close proximity to each other in all three directions of the mass, so that the mass is subjected to the magnetizing action of the magnetic field (for example, a magnetic field obtained by a magnet having a strength of about 10,000 Gauss). When placed on the order of 30 seconds or less and for a short period of time, preferably 10 seconds or less, particles in close proximity are magnetized.
[0016]
Experiments have shown that this magnetization gradually decays for at least 20-30 minutes, and in some cases, decays to a point where it disappears. The magnetization described above is referred to herein as “non-permanent collective magnetization” or “residual magnetization”. This non-persistent collective magnetization or “residual magnetization” is preferably measured within a set time in minutes excluding the influence of the magnetic field from the mass.
[0017]
By normalizing this interval to 5 minutes during the study underlying the present invention, particles having the same identity characteristics (ie, particle diameter, surface treatment, and the identity of the biomolecule to which it binds). It has been found that a standard curve can be constructed and reacts with these particles, thus easily calculating the number or concentration of target molecules present in the test sample.
[0018]
In these experiments, individual scattered particles of superparamagnetic material having the same average diameter range physical size and the same surface treatment are molecules that actually react with the target molecules of the test sample. It was found that it did not retain measurable magnetization when the magnetic field was removed, regardless of whether it bound first or not to the same biomolecule. Conclusion that because they did not retain magnetization, it was not necessary to physically separate them before measuring the non-permanent collective magnetization of the massive interaction mass of superparamagnetic particles and bioorganic molecules Reached. This is theoretical because the latter results in measurable magnetic recording and its sensitivity to thermal effects is low. Non-reacting superparamagnetic labeled antibodies or other biomolecules have a smaller physical particle size compared to superparamagnetic labeled immune complexes or other superparamagnetic labeled reaction biomass. The superparamagnetism of labeled antibodies or other biomolecules and the direction of their magnetization are easily dissipated immediately as a result of thermal effects, and the direction of magnetization in superparamagnetically labeled antibodies is very rapid, as is the case with superparamagnetic particles alone. Are thought to tend to be random.
[0019]
The initial experiments described and discussed in this specification and the parent application were performed by an experimental prototype measuring instrument, the Ericomp Maglab 2000, which is currently on the market as far as the applicant knows from knowledge and information. Is not out. This application adds a discussion and explanation of similar studies conducted using SQUID as the measuring instrument. This study shows that superparamagnetic particles bound to a conjugate of a biomolecule-binding pair, such as an antibody-antigen conjugate, further bind the biomolecule-paired superparamagnetic particles to form a sandwich, eg, tagged A non-binding paramagnetic particle, and a single biomolecule in that it forms an antibody-antigen-antibody sandwich and in that it retains measurable magnetization in that form beyond that period It behaved differently from the particles forming the binder. In contrast, superparamagnetic particles that do not bind and a “sandwich” of superparamagnetic particle tag biomolecule-biomolecule binding partner-immobilized biomolecule present in excess or otherwise (superparamagnetic particle tag antibody-antigen A molecule bound to a single biomolecule that cannot form a fixed antibody (such as a “sandwich”) does not retain magnetization, and thus, for example, a tagged antibody-antigen-fixed antibody sandwich The superparamagnetic particle tag present in the structure is easily separated by simply washing it from a cross-linked structure that exhibits a retained magnetization for a measurable period.
[0020]
Similarly, in experiments utilizing the SQUID newly described herein, an immunochromatographic cell is formed at one end of which a tagged antibody-antigen-fixed antibody sandwich is formed as described hereinafter. On the strip, the magnetization of the superparamagnetic tag in these sandwich structures is longer when the strip is moistened than when the strip is first dried and then measured for its magnetization, and may persist at a higher intensity. found. This latter finding was investigated several times and was unexpected.
[0021]
In the method according to the present invention, (1) (a) binding or adsorbing the same biomolecule which is a member of a biological binding pair to each group of superparamagnetic particles,
(B) the product of step (a) on a sample selected from a liquid and a solid that contains or is suspected of containing a biological binding partner of a biomolecule bound or adsorbed to a superparamagnetic particle Contacting the stage,
(C) The superparamagnetic particle-biomolecule conjugate or superparamagnetic particle-biomolecule adsorbate from step (a) is reacted with any biobinding partner molecule present in the sample to bind to the linked biomolecule. Forming a complex and tightly coupled three-dimensional mass comprising the superparamagnetic particles produced,
(D) after exposing the mass to the magnetic field for the shortest time necessary to induce the magnetization of the superparamagnetic particles in the mass, immediately removing the magnetic field, thereby causing the superparamagnetic particles in the mass to Cooperatively showing measurable non-persistent collective magnetization lasting at least 20 minutes after exposure, and any of the following steps:
(E) confirming the presence of such magnetization by suitable equipment if only qualitative results are desired, or
(F) the biomolecule of step (a) or step (b), which measures the magnetic signal intensity of the non-permanent collective magnetization before it dissipates and which is involved in the formation of the mass as described in step (c) A method for detecting a biological reaction, comprising the step of correlating to one quantitative concentration or number.
[0022]
In the method according to the present invention, (2) Fe having an average diameter of 1 nm to about 100 nm when the superparamagnetic particles are measured by X-ray diffraction and a transmission electron microscope.₃O₄The method according to (1), comprising:
[0023]
In the method according to the present invention, (3) each superparamagnetic particle binds to an antibody, and the sample in step (b) is a liquid sample containing an antigen that is a specific binding partner of the antibody, Is obtained by executing step (f), characterized in that it is a method according to (2).
[0024]
In the method according to the present invention, the period of exposure to the magnetic field in (4) step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and a transmission electron microscope is 5 nm to 60. The method according to (3), which is in the range of nm.
[0025]
The method according to the present invention is characterized in that (5) the method is described in (4), which is an immunoassay method in which the antigen content of the sample is quantified in step (f).
[0026]
The method according to the present invention is characterized in that it is a method according to (5), which is carried out in (6) side channel type.
[0027]
The method according to the present invention is characterized in that it is a method according to (6), which is carried out in the form of (7) immunochromatography assay.
[0028]
The method according to the present invention is characterized by (8) the method according to (5), which is carried out in a vertical flow or flow-through type.
[0029]
In the method according to the present invention, (9) a superparamagnetic particle is composed of a single magnetizable metal superparamagnetic particle, two combined magnetizable metal superparamagnetic particles, or a single magnetizable metal or two combined The method according to (1), comprising particles selected from superparamagnetic particles of an oxide of one of two magnetizable metals.
[0030]
In the method according to the present invention, (10) each superparamagnetic particle binds to an antibody, and the sample of step (b) is a liquid sample containing an antigen that is a specific binding partner of the antibody; The method according to (9), wherein a quantitative result is obtained by performing f).
[0031]
In the method according to the present invention, the period of exposure to the magnetic field in (11) step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and a transmission electron microscope is 5 nm to 50. The method according to (10), which is in the range of nm.
[0032]
The method according to the present invention is (12) the method according to (11), which is an immunoassay method in which the antigen content of the sample is quantified in step (f).
[0033]
The method according to the present invention is the method according to (12), wherein the method is performed in (13) side channel type.
[0034]
The method according to the present invention is characterized in that it is a method according to (13), which is carried out in the form of (14) immunochromatography assay.
[0035]
The method according to the present invention is characterized by (15) the method according to (12), which is carried out in a vertical flow or flow-through type.
[0036]
In the method according to the present invention, (16) superparamagnetic particles having a spinel structure determined by X-ray diffraction analysis and a transmission electron microscope include superparamagnetic particles of two magnetizable metal oxides combined. (9) The method is described.
[0037]
In the method according to the invention, (17) each superparamagnetic particle binds to an antibody, the sample of step (b) is a liquid sample containing an antigen that is a specific binding partner of the antibody, and The method according to (16), wherein a quantitative result is obtained by performing f).
[0038]
In the method according to the present invention, the period of exposure to the magnetic field in (18) step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and a transmission electron microscope is 5 nm to 50. The method according to (17), which is in the range of nm.
[0039]
The method according to the present invention is (19) the method according to (18), which is an immunoassay method in which the antigen content of the sample is quantified in step (f).
[0040]
The method according to the present invention is the method according to (19), wherein the method is carried out in (20) side channel type.
[0041]
The method according to the present invention is characterized in that it is a method according to (20), which is carried out in the form of (21) immunochromatographic assay.
[0042]
The method according to the present invention is (22) the method according to (19), which is carried out in a vertical flow or flow-through type.
[0043]
The method according to the present invention is characterized in that (23) the same biomolecule is adsorbed to superparamagnetic particles in step (a).
[0044]
In the method according to the present invention, the period of exposure to the magnetic field in (24) step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and a transmission electron microscope is 5 nm to 50. It is the method of (23) description which is the range of nm.
[0045]
The method according to the present invention is characterized in that it is the method according to (24), which is (25) an immunoassay.
[0046]
The method according to the present invention is the method according to (25), wherein the method is performed in (26) side channel type.
[0047]
The method according to the present invention is characterized in that it is a method according to (26), which is carried out in the form of (27) immunochromatographic assay.
[0048]
The method according to the present invention is (28) a method according to (26), which is carried out in a vertical flow or flow-through type.
[0049]
In the method according to the present invention, (29) the superparamagnetic particles are any of superparamagnetic particles, superparamagnetic particles of two magnetizable metals combined, or a single magnetizable metal or two magnetizable metals combined. It is the method of (23) description including the particle | grains selected from the superparamagnetic particle | grains of these oxides.
[0050]
In the method according to the present invention, (30) superparamagnetic particles having a spinel structure determined by X-ray diffraction analysis and transmission electron microscope include superparamagnetic particles of two magnetizable metal oxides combined. (23) It is the method of description.
[0051]
The method according to the present invention is characterized in that (31) step (f) is performed in place of step (e).
[0052]
In the method according to the present invention, as referred to in step (b) described in (32) (1), the method is repeated for a series of samples each containing different concentrations of a given biological binding partner. Thus, the measurement in step (f) of the magnetic signal intensity from the non-permanent collective magnetization of the mass described in step (d) described in (1) is the same as in steps (c) and (d) described in (1) The method according to (31), wherein the method is uniformly performed on each sample at the same time interval from the time when the exposure to the magnetic field of the mass described in each is removed.
[0053]
In the method according to the present invention, as described in step (b) described in (33) (1), the concentration or number of molecules of a predetermined biological binding partner, and as described in (32) By measuring the signal in step (f) at a uniform time interval with respect to the resulting series of samples, non-permanent of the mass containing it as described in step (c) and (d) as described in (1) Once the correlation is established between the magnetic signal strength of the aggregate magnetization and whenever the method described in (31) is performed on any sample containing an unknown concentration or number of molecules of the same biological binding partner, the step ( The method according to (31), wherein the same uniform time interval is adhered to in f).
[0054]
The method according to the present invention is (34) the method according to (1), wherein the average diameter of the superparamagnetic particles measured by X-ray diffraction and a transmission electron microscope is in the range of 1 nm to 60 nm. To do.
[0055]
In the method according to the present invention, (35) (a) a step of binding or adsorbing the same biomolecule which is a member of a biological binding pair to each of the group of superparamagnetic particles,
(B) the product of step (a) on a sample selected from a liquid and a solid that contains or is suspected of containing a biological binding partner of a biomolecule bound or adsorbed to a superparamagnetic particle Contacting the stage,
(C) The superparamagnetic particle-biomolecule conjugate or superparamagnetic particle-biomolecule adsorbate from step (a) is reacted with any biobinding partner molecule present in the sample to bind to the linked biomolecule. Forming a complex and tightly bound three-dimensional mass comprising the superparamagnetic particles produced;
(D) immediately removing the magnetic field after exposing the mass to the magnetic field for the shortest period necessary to induce magnetization of the superparamagnetic particles in the mass; and
(E) A method for detecting a biological reaction, comprising the step of measuring the magnetic signal intensity of the mass at regular intervals for a certain period with a suitable device in order to detect the decay period.
[0056]
In the method according to the invention, (36) the product of step (a) is supported on one end of an immunochromatographic (“ICT”) strip, the strip is on the other end, and superparamagnetic in step (a). Providing a capture zone consisting of immobilized unbound biomolecules identical to the molecules adsorbed to the particles, and wherein the product comprises in step (b) a biological binding partner of the biomolecule of step (a) The liquid sample is brought into contact with the suspected liquid sample and the liquid sample picks up the product of step (a) and passes it along the strip in step (c) to the superparamagnetic particle tag biomolecule-biological binding partner. The step according to (35), wherein a flow is passed through the capture zone where a complex and tightly bound three-dimensional mass of fixed biomolecules is formed, a portion of the ICT strip containing the three-dimensional mass is cut from the strip; Wet during (d) and (e) It is maintained in the state, characterized in that it is a method of (35) wherein.
[0057]
In the method according to the present invention, (37) (a) each of the superparamagnetic particles is bound or adsorbed with the same biomolecule which is a member of a biological binding pair, and the strip is chromatographed on the strip. The resulting conjugate is collected in contact with a liquid sample that flows along and along the strip along with the liquid sample, near the end receiving the sample of the immunochromatographic (“ICT”) strip. Storing in position,
(B) adding a liquid sample known or suspected of containing a biological binding partner of the same biomolecule described in step (a) to the end receiving the sample of the ICT strip; Flow along the strip on chromatography, collect the mobilized and stored conjugate formed in step (a), and flow the sample and the conjugate together along the strip Forming additional conjugates of superparamagnetic particles-biomolecules-biological binding partners as they flow,
(C) contacting the fluid mass of step (b) with a pre-stored filament of immobilized biomolecules known to react with the biological binding partner and linking the biomolecule-biology Forming a complex and tightly bound three-dimensional mass along the striation comprising a biological binding partner reaction product and tightly bound superparamagnetic particles;
(D) separating the portion of the ICT strip containing the striations along which the tightly bound three-dimensional mass is formed from the rest of the strip in a wet state by cutting or other suitable mechanical means. Stage,
(E) maintaining the separated portion in a wet state and placing it under the influence of a magnetic field to induce magnetization of the superparamagnetic particle; and
(F) measuring the aggregate permanent magnetization of the particles at a desired interval or series of intervals while maintaining the magnetic constant and maintaining the debris in the wet state under the influence of the same magnetic field. It is the method of detecting.
[0058]
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, superparamagnetic particles that are individually too small to maintain any degree of remanent magnetization can be applied to a magnetic field of as short as possible, preferably 10 seconds or less, and no more than 30 seconds, with a strength of about 10,000 Gauss. Has been shown to acquire a measurable non-permanent collective magnetization, ie, a labeled superparamagnetic antibody-antigen-fixed antibody “sandwich” mass, a labeled platelet clot Acquiring the collective magnetization of three-dimensional biomass that interacts together when incorporated into a densely packed three-dimensional mass, such as protein mass separated by chromatography, etc. It is shown.
[0059]
The use of superparamagnetic particles as labels for biological reactions, including biochemistry, provides substantial advantages over many labels currently used, for example, in various assay systems. For example, superparamagnetic particles, in contrast to ferromagnetic particles, do not exhibit remanent magnetization and do not exhibit magnetic properties until affected by a magnetic field. They are therefore virtually unlimited storage-stable as opposed to many commonly used labeling materials, including metal colloids, enzymes, chemiluminescent agents, radioactive trace elements, and the like.
[0060]
Because of their stability, they are easy to mix with other materials, float in liquids, and otherwise easy to handle unless exposed to a magnetic field of sufficient strength to excite magnetization.
[0061]
In the sense of the present invention, as observed, non-persistent collective magnetization is a measurable phenomenon that is a linear function of the concentration or number of target biomolecules in a test sample. However, a series of tests, i.e. tests of target analyte molecules at different concentrations, tests to generate a standard curve, with the intention to rely on a standard curve already created to determine the concentration present in the sample In order to obtain an equivalent result in a test or the like, care must be taken to measure the non-permanent collective magnetization at the same time interval after removing the magnetic field that induces this magnetization.
[0062]
The size of the particles, the surface characteristics of the particles, the strength of the magnetic field, and the time taken for the magnetization phase are the average distance between the magnetized particles that are captured in the end product mass of the bioorganic material and the particles that are bound, as well as non-permanent It is thought that it will have some role in the length of time it lasts and the rate at which it decays. Similarly, the measurement of the overall magnetization is not limited to any other uniform time interval from the magnetization phase (Example 5 and the 5-minute interval after the magnetization phase selected in other studies in which the ellicomp instrument is utilized, as well as the SQUID instrument). In a shorter period of time, such as selected in studies involving), at least in the systems examined so far, the decay will be at a rate that can provide a correlation between the number of bound particles and the concentration of the target analyte or other target molecule. Seems to happen. In measuring the overall magnetization, depending on the “platform” or biological matrix that may be present, measurements are made at intervals that result in recordings that greatly exceed any background magnetization measurements that need to be estimated. So be careful. Furthermore, before choosing different intervals for the overall magnetization measurement, it is necessary to ensure that the decay rate of the non-permanent collective magnetization follows a consistent pattern of superparamagnetic particles that have undergone the same procedure. is there.
[0063]
As used herein, “superparamagnetic particles” are magnetizable when closely packed together in a mass of interacting bioorganic materials, but do not retain residual magnetism. , And particles that do not exhibit remanence when measured individually after attempting magnetization. These particles are pure metals such as iron, cobalt, cadmium, zinc, magnesium, manganese, etc., known to be easily magnetizable, iron oxide, CoFe₂O₄MgFe₂O₄And other metal oxides known to be easily magnetized when placed in a magnetically affected place such as iron, zinc, nickel, cadmium, cobalt, etc. . For example, iron and its oxides and blends with other metals and their oxides, which exhibit a spinel structure when examined by X-ray diffraction and transmission electron microscopy, can be used as well. It should be noted that a pure metal is superparamagnetic only if the average diameter is within a physical size range limited to a few nanometers. Pure metal superparamagnetic particles are chemically unstable as well, but the corresponding superparamagnetic particle oxides are relatively inert and exhibit their superparamagnetism within a wider physical size range. maintain.
[0064]
These superparamagnetic particles are not inherently reactive with bioorganic materials, and often are desirable to bind target molecule binding partners that should be monitored, assayed, or otherwise localized and quantified. Coated with the substance to be reacted. Various methods and materials for such coatings have been used in the past to coat polymers or glasses, including glass beads and solid phase polymers, including inserts or “dipsticks” used in various assay systems. It is used. The same coating methods and materials are useful for coating superparamagnetic particles that are used to detect end products of biological reactions, including biochemistry. Various adsorption methods in which proteins or the like are adsorbed directly onto, for example, iron oxide are also well known and may also be utilized in the present invention to improve particle reactivity.
[0065]
Superparamagnetic particles are distinguished from ferromagnetic materials, including particles that acquire permanent remanent magnetization when exposed to an external magnetic field, and paramagnetic materials that have a positive magnetic susceptibility that is 0.001 times weaker than ferromagnetic materials. Is done. The magnetic susceptibility of superparamagnetic materials exists between ferromagnetic and paramagnetic materials, and the particles are referred to herein as intermediates between ferromagnetic and paramagnetic particles. Superparamagnetic systems show a slow relaxation time, i.e., they slowly return from a magnetized state to a non-magnetized state. The central behavioral supermagnetic relaxation time is the average time required for particle relaxation to jump from one direction to another. The particles used in this study, which is the basis of this application, had an average diameter of 5-15 nm as measured by X-ray diffraction and transmission electron microscopy. They are pure Fe with a spinel structure, as confirmed by X-ray diffraction and transmission electron microscopy.₃O₄Consisted of.
[0066]
It is known to physicists that superparamagnetic materials do not exhibit remanent magnetization. The hysteresis loop of superparamagnetic material (ie, the plot obtained by plotting magnetization against magnetic field strength) is curvilinear and is typically similar to that shown in FIGS. 2 and 2A. This is in contrast to the typical hysteresis loop of ferromagnetic materials (FIG. 1) and the linear hysteresis plot obtained for paramagnetic materials. Most commonly, superparamagnetic particles have a small average diameter on the order of less than 50 nm, often 30 nm or less, as measured by X-ray diffraction and transmission electron microscopy, In this system, larger particles with superparamagnetic properties are observed. (All of its size and behavior after removal from the magnetic field is epitomized by the suggestion that particles of 800 nm and larger are ferromagnetic and not superparamagnetic in the Adelmann et al. Paper. ing). Still further, it is typical for superparamagnetic particles that the magnetizations they can exhibit when subjected to a magnetic field decay with time until they completely dissipate. Finally, superparamagnetic particles have some degree of magnetic ordering, i.e. they are called subdomain structures consisting of a group of diverse sizes containing several atoms with unpaired electrons in an unmagnetized state However, the population is a “domain structure” of a ferromagnetic material characterized by a larger population called a magnetic domain in which each atom or other structural unit has an unpaired electron that imparts a true magnetic moment. Small and sparse compared to. In these latter materials, each magnetic domain exhibits a directional magnetic action that is the sum of all unpaired electron vectors present in that magnetic domain.
[0067]
In summary, ferromagnetic materials have strong magnetic ordering and superparamagnetic materials have some magnetic ordering but are much weaker than ferromagnetic materials. The presence of magnetic ordering in superparamagnetic materials has been confirmed by neutron diffraction measurements. Chen et al., “Superparamagnetic MgFe₂O₄Synthesis by superprecipitation of nanoparticles (Synthesis of Superparamagnetic MeFe₂O₄  See "Nanoparticulates by Replication" (J. Magnetism and Magnetic Materials, 94, 1-7 (1999)). Paramagnetic materials do not have magnetic ordering.
[0068]
For a good technical explanation of the similarities and differences when physicists recognize “ferromagnetic”, “superparamagnetic”, and “paramagnetic” materials, see Chen et al., “Mg₂FeO₄Size-Dependent Superparamagnetic Properties of Mg of Spinel Ferrite Nanocrystals (Size-Dependent Superparamagnetic Properties of Mg₂FeO₄  Spinel Ferrite Nanocrystals) ", Appl. Phys. Letters. 73, 3156-8 (1998).
[0069]
The ability to measure non-persistent collective magnetization in aggregated three-dimensional reaction products containing bioorganic materials closely associated with superparamagnetic particles may be attributed to the relatively stable macrostructure of these reaction products. The macrostructure holds the entrained particles in place and sustains the decay of magnetization imparted by exposure to a magnetic field. However, Applicant has not established this or any other scientific explanation for the reproducible phenomenon found in experimental studies relating to the present invention and is therefore bound by any particular explanation. Not intended.
[0070]
The present invention substantially includes in vitro biological reactions, including at least immunoassays, DNA probes, oligonucleotide probes, chromatographic separation of molecules, and other biological reactions desirable for quantifying the amount of target molecule present. Provides unlimited spectral enhancement. It is believed that the present invention may also be useful when monitoring certain in vivo biological responses. The detection system of the present invention comprises immunoassays in general, particularly immunochromatography and other “side channel” assays and so-called “flow-through” assays, ie, assays that include a vertical flow step in which reactants are carried together. Useful in law.
[0071]
In the October 2000 newsletter of Midwest Scientific Co., Shark Bytes describes the type of assay currently under development at Ohio State University. The method comprises a compact disc ("CD") that is rotated by a compact disc player with a small reservoir and channel to mix a medical sample suspected of containing the target analyte with a small pool of test reactants. Very useful to be quickly implemented and evaluated by the computer that will be included in the Ohio CD player system, including the superparamagnetic particle tag binding partners of specimens suspected of being in small pools on such test platforms A simple assay would be obtained. The computer can easily be programmed to read non-persistent collective magnetization imparted by an external magnetic field in digital form and correlate this record with stored information corresponding to a standard curve. Those skilled in immunoassay will recognize that the combination of an associated CD player computer and one CD, for example, by providing different antibodies for different target analytes bound to superparamagnetic particles present in different “pool” regions of the CD. It will be appreciated that several assays can be readily adapted to be performed simultaneously on a portion of a single test sample.
[0072]
In addition to CD, other “platform” materials that may allow superparamagnetic particle-tagged biomolecules to react with target biomolecules in a test sample are considered useful in studies conducted within the scope of the present invention. In addition to what the specific examples below show, examples that may have been specifically investigated in preliminary studies are balsa wood and glass. Using balsa wood, even if the material is essentially non-magnetic and non-magnetizable, its capillarity provides a record of non-persistent collective magnetization that exhibits a very large standard deviation. There is a possibility that. Fill these capillaries with non-magnetized plastics or bovine serum albumin, other proteins, polyethylene glycol, or other substances well known to block capillaries in “dipstick” type immunoassay devices described in the prior art The balsa tree would then become more acceptable as a platform. Glass turns out to be a satisfactory platform material without capillary problems as long as a suitable background record is obtained, and most glass slides can be magnetized to a low degree when exposed to a magnetic field. Can compensate for the fact that it contains enough iron. For this reason, when a biological reaction in which one reactant is labeled with superparamagnetic particles according to the present invention is performed on a glass platform, it is necessary to determine the background signal and subtract it from the sample record.
[0073]
Various instruments may be used to measure non-persistent collective magnetization. In this regard, several different research and development groups are developing relatively low cost measuring instruments that apply knowledge gathered from high resolution magnetic recording technology and computer disk drive technology. One of these is the Oerlik Comp Maglab 2000, at least one early prototype of which is described in the article of Adelman, J. Assn. For Lab. Automation cited earlier. The other is the Quantum Design, Inc. instrument described in US Pat. No. 6,046,585 issued April 4, 2000. Another Quantum Design, Inc. instrument is described in International Publication No. 01/40790, published June 7, 2001.
[0074]
The following specific examples are provided in colloidal gold in an immunochromatographic (“ICT”) assay for canine filamentous worms sold by the representative of the present patent application, Binax, Inc. The use of the superparamagnetic label according to the present invention instead of is described.
[0075]
【Example】
Example 1-Selection of coating agent for superparamagnetic particles
Superparamagnetic particles can be coated with a reagent having a free carboxyl functional group so that it can be covalently coupled to a specific antibody, such as an antibody against canine filamentous worms ("CHW"). Therefore, initial studies were conducted to identify the coating material selected for this purpose.
[0076]
Why rheological properties are important for the successful operation of ICT assays, and past experience with colloidal gold labels has shown that smaller particles are superior in rheology From the 10 nm diameter Fe₃O₄Particles were selected for this study. Their size was confirmed by X-ray diffraction and transmission electron microscopy.
[0077]
Using the coating method described in US Pat. No. 5,547,682, two polymer coating materials were examined for different lots of superparamagnetic particles. One lot of these particles was coated with chondroitin sulfate A (“CSA”) and the other lot was coated with small polyacrylic acid (“sPAA”).
In each case, the suspension of coated particles was 40-60 nm in diameter as measured by dynamic light scattering. Further testing of both CSA-coated and sPAA-coated superparamagnetic particles showed that the CSA-coated particles were excellent in stability and ability to bind to antibodies. The hysteresis loop measured by the vibrating sample magnetometer (“VSM”) test for three sets of particles, all of which are sPAA coatings and having different iron concentrations bound with the commercially available antibody betil, is shown in FIG. These uncoated particles are the same as the particles that were the starting material of Example 3. In FIG. 2, the shape of a typical hysteresis curve for superparamagnetic materials is shown, as well as confirming that these particles do not exhibit residual magnetization. FIG. 2A shows superparamagnetic 10 nm Fe by SQUID instrument (black square curve) and by VSM (white circle curve).₃O₄The hysteresis measurement performed about the coating particle of this is shown. Similarly, a plot of hysteresis measurements made by VSM of the same coated particles bound with a carboxypolysaccharide antibody is also shown in FIG. 2A. In all three, the hysteresis behavior of typical shaped superparamagnetic materials is shown, confirming that the particles do not exhibit residual magnetization.
[0078]
Example 2 Preparation of Superparamagnetic Particle Labeled Antibody
The CSA-coated superparamagnetic particles prepared in Example 1 were covalently coupled to the same anti-canine filamentous (“CHW”) antibody as the anti-CHW antibody used in the manufacture of the commercial VINAX ICT test for CHW antigen. The suspension was suspended in phosphate buffered saline containing 10 mg / ml bovine serum albumin until used in Example 3.
[0079]
Example 3-Using a superparamagnetic label CHW of ICT Performing the assay
Nitrocellulose ICT flow path test strips were treated in the same way as the strips used in the ICT assay sold by Vinax for CHW. These strips were assembled into a dipstick-type device by laminating to the opposite end adsorbent bed components of the flow path test strips. A series of solutions containing CHW antigen were prepared at antigen concentrations ranging from 100 pg / ml to 200 ng / ml. 190 μl of each of these antigen solutions was dispensed into different wells of a new polystyrene 96-well microtiter plate. To these samples, 5 μl of the CSA-coated superparamagnetic particle-labeled anti-CHW antibody of Example 2 was added. The labeled antibody / antigen solution mixture was incubated at room temperature for 15 minutes. Immediately following this incubation, the sample receiving end of the dipstick device was added to each labeled antibody / antigen solution mixture and the mixture was allowed to flow into the capture zone. The immobilized unlabeled rabbit polyclonal anti-CHW antibody that binds to the strip in the capture zone then reacts with the antigen-superparamagnetically labeled antibody conjugate, and the immobilized antibody-antigen-superparamagnetically labeled antibody “sandwich” enters the capture line. Form along. ICT strips were removed from the ICT apparatus 15 minutes later and each exposed to a 10,000 Gauss magnetic field for 10 seconds. Five minutes after removing the magnetic field, each strip was placed in the Oerlik Comp Maglab 2000 instrument so that the capture line could be seen in the detector field of view, and its non-permanent collective magnetization was read.
[0080]
Each antigen solution was tested in duplicate in the ICT test as described. Non-permanent collective magnetization records for both series of samples having a known concentration of about 1 ng / ml and higher are graphed against antigen concentration in FIG. In a series of 2 samples per test, the Fe content of the immune complex at the capture line of the ICT device used was the chemical calorific value using a commercially available ferritin test from Sigma Chemical Co. Determined by measurement method. As shown in FIG. 4, the results of the calorimetric chemical test and the recording of the magnetization are Fe₃O₄As well as a plot of iron concentration calculated in μg / ml.
[0081]
In the ICT test as performed in this example, any labeled antibody initially stored with a channel threshold that does not react with the antigen in the sample passes through the capture zone and is another pad upstream from that zone. Flowing to the position. These unreacted paramagnetic particle-labeled antibodies were subjected to the action of a magnetic field of 10,000 gauss for 10 seconds, left for 5 minutes and then placed in the sensor area of the Oerlik Comp Maglab 2000 instrument. It turns out not to show.
[0082]
From the results of the previous examples, the relationship between magnetic recording in relative magnetic units (“RMU”) and antigen concentration (target analyte) is essentially in the range of 1 ng / ml of antigen to 150 ng / ml of antigen. It was determined to be a linear function. See FIG. 3, a plot of relative magnetic units against antigen concentration ng / ml for each of the two series of CHW assays performed. However, instrument noise caused a large standard deviation in the recording of samples with antigen concentrations below 1 ng / ml. This is shown in the plot of measured relative magnetic units versus antigen concentration in pg / ml in FIG. The ICT strip with added 200 pg / ml of antigen is incorporated into a labeled antibody-antigen-fixed antibody sandwich where the antigen is recovered in the mass, subjected to a magnetic field of 10,000 gauss for 10 seconds and then left for 5 minutes. The fact that they showed a non-persistent collective magnetization that could be recorded nevertheless showed that the sensitivity of the test was significantly enhanced by using superparamagnetic labels instead of colloidal gold labels. ing.
[0083]
In an instrument with improved noise reduction, the physical sensitivity of the described superparamagnetic label is Fe₃O₄Fe 0.1 ng calculated as or about 10^-18Obviously, the wide dynamic sensitivity range is about 1-10.⁶It enters between relative units, which are probably highly resistant to interference from various biological matrices that may be present.
[0084]
Example 4
A dipstick type device was prepared as described in the first to third sentences of Example 3. A solution containing 100 ng / ml, 150 ng / ml and 200 ng / ml of canine filamentous worm antigen, which is a target antigen of the commercially available binax assay method for canine filamentous worms, was prepared to prepare a new polystyrene microtiter plate. Distribute into individual wells. To each sample, 5 μl of CSA coated superparamagnetic particle labeled anti-CHW antibody was added as described in Example 2. After each of the resulting labeled antibody / antigen mixtures was incubated at room temperature for 15 minutes, the end receiving the sample of the dipstick device was added to each well and chromatographed in its capture zone along the dipstick strip. The mixture was applied to form a “sandwich” of superparamagnetic labeled antibody-antigen-fixed antibody composition along the capture line in the capture zone. After 15 minutes, the dipstick device is removed from the well and the portion of each ICT piece containing all of the dipstick device's capture line is cut and dried, and 1 Tesla (“T”) (ie, “T”) in the SQUID (ie (10,000 Oersted, also called “10,000 Gauss”) for 10 seconds. Next, the magnetic field was switched off, and after 3 and a half minutes, the magnetic signal of each part was measured. The premise of the experiment is that all the magnetic moments of superparamagnetic nanoparticles, including particles bound to the antibody-antigen-immobilized antibody "sandwich" formed in the capture line, are juxtaposed in the direction of the magnetic field by the applied magnetic field. Met. When the magnetic field is switched off, the particles that are not bound to the “sandwich” quickly return to their original non-arranged state, whereas the particles that are bound to the connected structure of the sandwich return slowly. The 5.0 minute time interval until recording corresponds to a time-lag to measurement in an experiment with an Oerlikon instrument.
[0085]
The results of these experiments are shown in Table 1:
[Table 1]

“Magnetic recording” is an electromagnetic unit (emu).
[0086]
Another series of experiments was done exactly the same except that the first recording was made at 3.5 minutes, where 3.5 minutes was the time required for the SQUID field to return from 1 Tesla to 0. After this, the second recording was performed at 4.0 minutes, and the third recording was performed at 4.5 minutes. The results are shown in Table 2, and the blank indicates that no record was left.
[Table 2]

[0087]
Table 3 shows the results of another series of experiments where the antigen levels in the original solution were 50 ng / ml, 100 ng / ml and 200 g / ml, respectively. In all other aspects, this series of experiments was performed exactly the same as the experiments shown in Table 2.
[Table 3]

[0088]
A further series of experiments recorded (1) that the antigen level of the solution was 1 ng / ml, 10 ng / ml, and 200 ng / ml, respectively, and (2) the wet part instead of the dry part. The experiment was performed in the same manner as the experiments obtained in Tables 2 and 3, except that The results are shown in Table 4.
[Table 4]

[0089]
Example 5
Many instruments currently available operate only with a constant magnetic field, which is first applied to magnetize the existing superparamagnetic particles and must remain during the measurement, so throughout the test It was decided to conduct parallel experiments similar to those shown in Tables 1, 2, 3, and 4 of Example 4 with the SQUID maintained at a constant magnetic field of 1 Tesla. Table 1A below shows the records obtained under a constant magnetic field of 1 Tesla for samples prepared identically to the corresponding antigen concentration samples in Table 1.
These samples were measured at 5.0 minutes after first exposing each sample to a magnetic field for 10 seconds, similar to the samples whose records are shown in Table 1:
[Table 1A]

[0090]
Table 2A shows the records obtained for a sample prepared identically to the sample of Table 2 where a constant magnetic field is maintained throughout. In this table, record # 1 was obtained at 3.5 minutes after first exposing each sample to a constant magnetic field for 10 seconds, while record # 2 was obtained at 4.0 minutes and record # 3 was 4 Obtained in 5 minutes.
[Table 2A]

[0091]
Table 3A shows the records obtained for a sample prepared identically to the samples in Table 3, and the records were obtained with the same magnetic field and spacing specified in Table 2A.
[Table 3A]

[0092]
Table 4A shows the records obtained under the same magnetic field for wet parts prepared exactly as the corresponding samples from which the records of Table 4 were obtained.
[Table 4A]

[0093]
In the various data shown in Example 4, the records in Table 4 obtained for each wetted part, each containing the trapping area of the ICT piece, show that the non-persistent collective magnetization is retained at a greater degree of strength over time and at least the SQUID When measured by instrument, these “wet portions” or “wet pieces” samples show a more pronounced dose dependency than the corresponding dry samples. This is more easily seen by comparing FIGS. 7 and 8, in which FIG. 7 graphs data for M, ie, electromagnetic units, against antigen concentration for dry strip records # 1 and # 2 in Table 1. And for the wet piece record # 1 in Table 4, the data is also shown in the graph.
[0094]
Surprisingly, the data of Example 5 obtained in the SQUID at a constant magnetic field also shows a much more significant dose-dependent response for the “wet part” or “wet piece” sample than the corresponding dry sample. Again, compared to FIG. 10 where the wet strip record from Table 4A is also graphically represented, the previously defined M versus antigen concentration is graphed for the dry strip records # 1 and # 2 of Table 1A. In the illustrated FIG. 9, it can be easily seen.
[0095]
These findings show that excellent performance can be obtained if the sample being examined is kept wet, particularly with respect to minimizing the signal to noise ratio and thus maximizing sensitivity. This phenomenon relates not only to non-permanent collective magnetization measured after magnetization and measurement after removal of the magnetic field, but also to more general measurement systems that use a constant magnetic field during the measurement.
[0096]
Although the present invention has been exemplified in the context of well-known immunodiagnostic systems specific to the antigen of the causative agent of the canine disease Dirofilaria immitis, the present invention provides a wide range of applications. The results of observation phenomena that were previously thought to be difficult or impossible to measure have been greatly improved, or accurate quantitative measurements have been made possible by immunochemistry and / or biology. It will be readily apparent to vendors. Accordingly, the scope of the invention should be construed as limited only by the appended claims.
[0097]
【The invention's effect】
The present invention provides a method for detecting the occurrence of a biological reaction using superparamagnetic particles or for quantifying the result.
[Brief description of the drawings]
The attached drawings are as follows:
FIG. 1 is a schematic representation of a hysteresis curve for a typical ferromagnetic material obtained from the literature. In the diagram, H represents the applied magnetic field in Oersted and σ represents the magnetization obtained therefrom. The saturation of magnetization is represented by σs, and the residual or magnetization remaining after removing the magnetic field is represented by σr. The reverse magnetic field required to make σr zero is represented by the coercive force Hc.
FIG. 2 is a superimposed plot of measured hysteresis for superparamagnetic particles coated with small polyacrylic acid and then reacted with a commercially available antibody called betil. Three such measurements consist of three different iron concentrations measured for the suspension of particles: 2.4 mg / ml (● on the plot), 1.0 mg / ml (■ on the plot), and 0 .3 on the same treated particles at 3 mg / ml (▲ on the plot). In the plot, M is emu (electromagnetic units) / cc ("cm³)), H represents the applied magnetic field in Oersted, and Hc to load the magnetization (from the zero line to the right) or to reverse the magnetization (from the zero line to the left) The coercive force required for this is expressed in Oersted. FIG. 2A shows superparamagnetic Fe coated polymer (ie small polyacrylic acid coating or chondroitin sulfate A coating).₃O₄Similar plots of overlapped hysteresis measurements using two different instrument measurement techniques on the particles, the particles before coating are identical to those used in the experimental studies described herein, and the hysteresis measurements The Fe sample bound to the CPS antibody by a vibrating sample magnetometer (“VSM”)₃O₄The superparamagnetic particles were used. In FIG. 2A, polymer-coated superparamagnetic Fe performed using a SQUID instrument.₃O₄Particle measurements are represented by ■; superparamagnetic Fe coated with polymer₃O₄The measurements performed on the particles are represented by ○ and superparamagnetic Fe₃O₄The measurement of particle-antibody conjugate is represented by ▲.
FIG. 3 is a plot of the measured magnetization of two series of superparamagnetic particle-antigen-fixed antibody sandwiches measured at the capture line of an ICT device, relative to the magnetic unit relative to antigen concentration ng / ml. It is. This type of ICT device can be any of Howard Chandler's US Patent Application No. 07 / 706,639, now US Pat. No. 6,168,956, or any of its various pending patents and applications. All of which are assigned to Smith Kline Diagnostics, Inc., which is the representative of the present invention for the purpose of immunoassay for a wide range of substances. Authorized exclusively. These data were obtained from the study described in Example 3.
FIG. 4 shows Fe of superparamagnetic particle labeling used in Example 3.₃O₄Is a plot of the Fe concentration calculated as μg / ml against the measured CHW antigen concentration ng / ml. This data was obtained from the study described in Example 3.
FIG. 5 is a plot of measured magnetization for relative magnetic units versus antigen concentration pg / ml. Similarly, data from the study described in Example 3 is embodied.
FIG. 6 shows superparamagnetic Fe used in the experimental studies described herein.₃O₄Superparamagnetic Fe after coating with particles (Pattern A) and one of the above two polymers₃O₄It is a X-ray-diffraction schematic which shows the result of having measured the particle size of particle | grains (pattern B) and overlapping.
7 is a plot of M in electromagnetic units versus antigen concentration for dried strip data from Records 1 and 2 (Sample Series 1 and 2) in Table 2 of Example 4. FIG.
8 is a plot of M in the same units versus antigen concentration using wet strip data from Record 1 series in Table 4 of Example 4. FIG.
FIG. 9 is a plot similar to FIG. 7 from record 1 and 2 series of dried piece data of the samples in Table 2A of Example 5.
FIG. 10 is a plot similar to FIG. 8 from the wet piece data of the record 1 series of the sample in Table 4A of Example 5. FIG.

Claims (37)

(A) binding or adsorbing to each group of superparamagnetic particles the same biomolecule that is a member of a biological binding pair;
(B) the product of step (a) on a sample selected from a liquid and a solid that contains or is suspected of containing a biological binding partner of a biomolecule bound or adsorbed to a superparamagnetic particle Contacting the stage,
(C) The superparamagnetic particle-biomolecule conjugate or superparamagnetic particle-biomolecule adsorbate from step (a) is reacted with any biobinding partner molecule present in the sample to bind to the linked biomolecule. Forming a complex and tightly coupled three-dimensional mass comprising the superparamagnetic particles produced,
(D) after exposing the mass to the magnetic field for the shortest time necessary to induce the magnetization of the superparamagnetic particles in the mass, immediately removing the magnetic field, thereby causing the superparamagnetic particles in the mass to Cooperatively showing measurable non-persistent collective magnetization lasting at least 20 minutes after exposure, and any of the following steps:
(E) confirming the presence of such magnetization with a suitable instrument if only qualitative results are desired, or (f) measuring the magnetic signal strength of the non-permanent collective magnetization before it dissipates. A method for detecting a biological reaction comprising the step of correlating it with one quantitative concentration or number of biomolecules of step (a) or step (b) involved in the formation of the mass described in step (c) . The method of claim 1, wherein the superparamagnetic particles comprise Fe ₃ O ₄ having an average diameter of 1 nm to about 100 nm as measured by X-ray diffraction and transmission electron microscopy. Each superparamagnetic particle binds to an antibody, and the sample of step (b) is a liquid sample containing an antigen that is a specific binding partner of the antibody, and quantitative results are obtained by performing step (f). The method of claim 2, wherein: The period of exposure to a magnetic field in step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and transmission electron microscope is in the range of 5 nm to 60 nm. Method. 5. The method according to claim 4, which is an immunoassay, wherein the antigen content of the sample is quantified in step (f). 6. The method of claim 5, wherein the method is performed in a side channel format. 7. The method of claim 6, wherein the method is performed in the form of an immunochromatographic assay. 6. The method of claim 5, wherein the method is performed in a vertical flow or flow through format. A superparamagnetic particle is a single magnetizable metal superparamagnetic particle, a combination of two magnetizable metal superparamagnetic particles, or a single magnetizable metal or a combination of two magnetizable metal oxides. The method of claim 1, comprising particles selected from superparamagnetic particles. Each superparamagnetic particle binds to the antibody, the sample of step (b) is a liquid sample containing an antigen that is a specific binding partner of the antibody, and quantitative results are obtained by performing step (f). 10. The method of claim 9, wherein: The period of exposure to a magnetic field in step (d) is 5 to 10 seconds, and the average diameter of superparamagnetic particles measured by X-ray diffraction and transmission electron microscopy is in the range of 5 nm to 50 nm. Method. 12. The method according to claim 11, which is an immunoassay, wherein the antigen content of the sample is quantified in step (f). 13. The method of claim 12, wherein the method is performed in a side channel type. 14. The method of claim 13, wherein the method is performed in the form of an immunochromatographic assay. 13. The method of claim 12, wherein the method is performed in a vertical flow or flow through format. The method of claim 9, wherein the superparamagnetic particles exhibiting a spinel structure as determined by X-ray diffraction analysis and transmission electron microscopy comprise superparamagnetic particles of two magnetizable metal oxides combined. Each superparamagnetic particle binds to the antibody, the sample of step (b) is a liquid sample containing an antigen that is a specific binding partner of the antibody, and quantitative results are obtained by performing step (f). 17. The method of claim 16, wherein: The period of exposure to a magnetic field in step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and transmission electron microscope is in the range of 5 nm to 50 nm. Method. The method according to claim 18, wherein the antigen content of the sample is quantified in step (f). 20. A method according to claim 19 performed in a side channel type. 21. The method of claim 20, wherein the method is performed in an immunochromatographic assay format. 20. The method of claim 19, wherein the method is performed in a vertical flow or flow through format. The method of claim 2, wherein the same biomolecule is adsorbed to the superparamagnetic particles in step (a). 24. The period of exposure to a magnetic field in step (d) is 5 to 10 seconds, and the average diameter of the superparamagnetic particles measured by X-ray diffraction and transmission electron microscopy is in the range of 5 nm to 50 nm. Method. The method according to claim 24, which is an immunoassay. 26. The method of claim 25, wherein the method is performed in a side channel format. 27. The method of claim 26, wherein the method is performed in an immunochromatographic assay format. 27. The method of claim 26, wherein the method is performed in a vertical flow or flow through format. The superparamagnetic particles are selected from superparamagnetic particles, superparamagnetic particles of two magnetizable metals combined, or superparamagnetic particles of an oxide of either a single magnetizable metal or two magnetizable metals combined. 24. The method of claim 23, comprising particles. 24. The method of claim 23, wherein the superparamagnetic particles exhibiting a spinel structure as determined by X-ray diffraction analysis and transmission electron microscopy comprise two magnetizable metal oxide superparamagnetic particles combined. The method of claim 1, wherein step (f) is performed instead of step (e). A method, as referred to in step (b) of claim 1, which is repeated for a series of samples each containing different concentrations of a given biological binding partner, comprising step (d) of claim 1 The measurement in step (f) of the magnetic signal intensity from the non-permanent collective magnetization of the mass described in claim 1 gives the exposure of the mass described in steps (c) and (d) of claim 1 to the magnetic field. 32. The method of claim 31, wherein the method is performed uniformly on each sample at the same time interval from the time removed. A uniform time interval for a series of samples obtained as defined in claim 32 and the concentration or number of molecules of a given biological binding partner as described in step (b) of claim 1 Once between the magnetic signal intensity of the non-permanent collective magnetization of the mass comprising it as described in steps (c) and (d) according to claim 1 by measuring the signal in step (f) If the correlation is established, the same uniform time interval is adhered to in step (f) whenever the method of claim 31 is performed on any sample containing an unknown concentration or number of molecules of the same biological binding partner. 32. The method of claim 31, wherein: The method according to claim 1, wherein the average diameter of the superparamagnetic particles measured by X-ray diffraction and a transmission electron microscope is in the range of 1 nm to 60 nm. (A) binding or adsorbing to each group of superparamagnetic particles the same biomolecule that is a member of a biological binding pair;
(B) the product of step (a) on a sample selected from a liquid and a solid that contains or is suspected of containing a biological binding partner of a biomolecule bound or adsorbed to a superparamagnetic particle Contacting the stage,
(C) The superparamagnetic particle-biomolecule conjugate or superparamagnetic particle-biomolecule adsorbate from step (a) is reacted with any biobinding partner molecule present in the sample to bind to the linked biomolecule. Forming a complex and tightly coupled three-dimensional mass comprising the superparamagnetic particles produced,
(D) exposing the mass to the magnetic field for the shortest period necessary to induce magnetization of the superparamagnetic particles in the mass, and immediately removing the magnetic field; and (e) the magnetic signal intensity of the mass, A method for detecting a biological reaction, comprising the step of measuring at regular intervals over a period of time with a suitable device to detect the decay period. The product of step (a) is supported on one end of an immunochromatography (“ICT”) strip, and the strip is immobilized on the other end, identical to the molecule adsorbed to the superparamagnetic particles in step (a). Providing a capture zone composed of unbound biomolecules and contacting the product in step (b) with a liquid sample suspected of containing a biological binding partner of the biomolecule of step (a); The sample picks up the product of step (a) and binds it along the strip in step (c) to the complex and tight binding of superparamagnetic particle tag biomolecule-biological binding partner-immobilized biomolecule 36. Flowing through the capture zone where a three-dimensional mass is formed, wherein a portion of the ICT strip containing the three-dimensional mass is cut from the strip and wetted during steps (d) and (e) of claim 35 36. maintained in a state. Method. (A) Each group of superparamagnetic particles binds or adsorbs the same biomolecule that is a member of a biological binding pair, is contacted with a liquid sample that flows along the strip on chromatography, and is collected Storing the resulting conjugate at a location near the end that receives the sample of the immunochromatographic (“ICT”) strip so that it flows along the strip along with the liquid sample;
(B) adding a liquid sample known or suspected of containing a biological binding partner of the same biomolecule described in step (a) to the end receiving the sample of the ICT strip; Flow along the strip on chromatography, collect the mobilized and stored conjugate formed in step (a), and flow the sample and the conjugate together along the strip Forming additional conjugates of superparamagnetic particles-biomolecules-biological binding partners as they flow,
(C) contacting the fluid mass of step (b) with a pre-stored filament of immobilized biomolecules known to react with the biological binding partner and linking the biomolecule-biology Forming a complex and tightly bound three-dimensional mass along the striation comprising a biological binding partner reaction product and tightly bound superparamagnetic particles;
(D) separating the portion of the ICT strip containing the striations along which the tightly bound three-dimensional mass is formed from the rest of the strip in a wet state by cutting or other suitable mechanical means. Stage,
(E) maintaining the separated portion in a wet state and placing it under the influence of a magnetic field to induce magnetization of the superparamagnetic particle; and (f) maintaining the magnetic field constant while maintaining the same magnetic field. A method of detecting a biological response comprising measuring the aggregate permanent magnetization of the particles at a desired interval or series of intervals while maintaining the debris in a wet state under influence.

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