JP2013061325A - Lelia (liposome-based enzyme-linked immunoassay) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new LELIA (Liposome-based Enzyme-Linked ImmunoAssay) capable of suppressing a background value and lowering a measurement limit compared with an ELISA (Enzyme-Linked ImmunoSorbent Assay).SOLUTION: An LELIA includes: a secondary antibody coupling process for reacting a primary antibody for recognizing a subject substance with antibody subject substance coupling liposome with which the subject substance is coupled in a liquid phase, adding an enzyme-labeled secondary antibody to be coupled with the primary antibody to the liposome and reacting the liposome with the primary antibody, and defining the liposome as antibody subject substance primary antibody enzyme-labeled secondary antibody coupling liposome; and a detection process for performing detection by adding a detectable substrate and performing a reaction with the enzyme-labeled secondary antibody.

Description

  The present invention relates to an enzyme immunoassay technique LELIA (Liposome-based Enzyme-Linked ImmunoAssay) using liposomes.

This inventor developed the technique regarding the recombinant proteoliposome for diagnosis for detecting the antibody contained in the blood (patent document 1). However, this technology is based on a protein having a domain that binds to and penetrates the double membrane that constitutes the liposome, and could not be used for the measurement of proteins and antibodies that do not have membrane-binding ability. .
On the other hand, an ELISA (Enzyme-Linked ImmunoSorbent Assay) method is known as a technique for measuring a very small amount of sample. The ELISA method is roughly classified into a direct adsorption method for measuring the antigen or primary antibody adsorbed on the solid phase and a sandwich method for recognizing the antigen with the primary antibody using the antibody adsorbed on the solid phase.
In the direct adsorption method, a solution sample containing an antigen is brought into contact with a solid phase (plate), and the antigen (containing substance) is immobilized on the plate surface to be immobilized. Next, a sample containing a primary antibody that recognizes the antigen is added to bind the antigen and the primary antibody. Next, an enzyme-labeled secondary antibody that recognizes the primary antibody is reacted to bind the antigen, the primary antibody, and the enzyme-labeled secondary antibody. Finally, the reactant is measured by adding a substrate and reacting with an enzyme. According to this method, the presence or absence of the antigen or the concentration of the primary antibody can be measured.
In the sandwich method, an antibody that reacts with an antigen is immobilized on a plate surface and immobilized in advance. The sample containing an antigen is added here, and an antibody and an antigen are combined. Next, a sample containing a primary antibody that recognizes the antigen is added to bind the antigen and the primary antibody, and the antigen is sandwiched between the two types of antibodies. Next, an enzyme-labeled secondary antibody that recognizes the primary antibody is reacted to bind the antigen, the primary antibody, and the enzyme-labeled secondary antibody. Finally, the reactant is measured by adding a substrate and reacting with an enzyme. According to this method, the concentration of the antigen can be measured.
The above ELISA method is used not only for research purposes but also for environmental analysis, food analysis, clinical diagnosis and the like. For example, ELISA is applied to a method for measuring blood insulin (Non-patent Document 1) and a method for measuring CRF (Corticotropin Releasing Factor) (Non-patent Document 2). According to these data, the measurement limit for insulin is 0.07 mU // L (0.42 pmol / L) and the measurement limit for CRF is 0.33 ng / mL.

WO 2007/094395

Mercodia AB [online] [retrieved on 2011-08-03] Retrieved from the Internet: <URL: http://www.funakoshi.co.jp/data/datasheet/MRD/10-1132-01.pdf> PHOENIX PHARMACEUTICALS, INC [online] [retrieved on 2011-08-03] Retrieved from the Internet: <URL: http: //www.phoenixpeptide.com/catalog/product_info.php? Products_id = 4247> Kanta Tsumoto, et al, Colloids and surfaces B: Biointerfaces 68 (2009) 98-105 McBride, J. D. and Cooper, M. A. J. Nanobiotechnology 2008, 6: 5. Helica Biosystems, Inc., [online] [retrieved on 2011-08-16] Retrieved from the Internet: <URL: http://www.helica.com> Ala-Kleme, T., Maekinen, P., Ylinen, T., Vaere, L., Kulmala, S., Ihalainen, P. and Peltonen, J. Anal. Chem. 2006, 78, 82-88. Zlatanovic, S., Mirkarimi, LW, Sigalas, MM, Bynum, MA, Chow, E., Robotti, KM, Burr, GW, Esener, S. and Grot, A. Sensors and Actuators B: Chemical 2009, 141, 13 -19. Chang, Y. S., Wu, C. H., Chang, R. J. and Shiuan, D. J. Biochem. Biophys. Methods 1994, 29, 321-329. Hou, S. Y., Chen, H. K., Cheng, H. C. and Huang, C. Y. Anal. Chem. 2007, 79, 980-985.

As described above, the ELISA method is a useful method having many applications. However, since the background value is high and the S / N ratio is low, there is a problem that the measurement limit cannot be lowered sufficiently.
The present invention has been made in view of the above problems, and its purpose is to provide a novel enzyme immunoassay technique using a liposome capable of suppressing the background value lower than the ELISA method and lowering the measurement limit. It is to provide LELIA (Liposome-based Enzyme-Linked ImmunoAssay).

Thus, the enzyme immunoassay method according to the first invention for achieving the above object includes (1) a covalent binding step in which a test substance is covalently bound to the surface of a liposome containing a sugar-containing lipid thin film to form a test substance-bound liposome. (2) A container adsorption step in which the test substance-bound liposome is added to the first container and adsorbed on the wall surface of the container, (3) a liposome washing step to remove the test substance-bound liposome that cannot be adsorbed, (4) liquid In the phase, the test substance-bound liposome and the primary antibody that recognizes the test substance are reacted to form a test substance / primary antibody-bound liposome, (5) test substance / primary antibody-bound liposome and liquid phase (6) In the liquid phase, in the test substance / primary antibody-binding liposome, in the primary antibody, A secondary antibody binding step of adding an enzyme-labeled secondary antibody to be reacted and reacting with the primary antibody to form a test substance / primary antibody / enzyme-labeled secondary antibody-binding liposome, (7) the test substance / primary antibody / enzyme label A secondary antibody washing step of separating the secondary antibody-bound liposome and the liquid phase and removing an unnecessary liquid phase portion; (8) adding the solution; and adding the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome After suspending, transfer the solution to the second container, and (9) adding a substrate that can be detected by enzyme reaction of the enzyme-labeled secondary antibody and reacting with the enzyme-labeled secondary antibody. And a detection step of performing detection.
According to the first invention, the test substance bound to the liposome surface can be detected and measured by the direct method (the test substance is directly recognized only by the primary antibody).

In addition, the enzyme immunoassay method according to the second invention includes (1) a covalent binding step in which an antibody recognizing a test substance is covalently bound to the surface of a liposome containing a saccharide-containing lipid thin film to form an antibody-bound liposome; In the first container, the antibody-binding liposome is adsorbed on the wall surface of the container, and (3) the liposome washing step for removing the non-adsorbable antibody-binding liposome; (4) in the liquid phase, the antibody A test substance binding step in which a binding liposome and a sample containing the test substance coexist to bind the antibody and the test substance to form an antibody / test substance binding liposome; (5) the antibody / test substance binding liposome; A test substance washing step of separating the liquid phase and removing an unnecessary liquid phase part; (6) in the liquid phase, the antibody / test substance-bound liposome bound with the test substance A primary antibody binding step in which a primary antibody that recognizes a substance is reacted to form an antibody / test substance / primary antibody-binding liposome; (7) an unnecessary liquid by separating the antibody / test substance / primary antibody-binding liposome from the liquid phase; (8) In the liquid phase, an enzyme-labeled secondary antibody that binds to the primary antibody is added to the antibody / test substance / primary antibody-binding liposome in the liquid phase, and reacted with the primary antibody. A secondary antibody binding step for preparing an antibody / test substance / primary antibody / enzyme-labeled secondary antibody-binding liposome; (9) separating the antibody / test substance / primary antibody / enzyme-labeled secondary antibody-binding liposome and liquid phase; (10) A solution is added, and the antibody / test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome is suspended, and this solution is added to the second antibody washing step. In the container And (11) a detection step of adding a substrate that can be detected by an enzyme reaction of the enzyme-labeled secondary antibody, reacting with the enzyme-labeled secondary antibody, and performing detection. .
According to the second invention, the test substance recognized by the antibody bound to the liposome surface can be detected and measured by the sandwich method (the test substance is sandwiched between the antibody and the primary antibody).
The first invention and the second invention are useful not only for quantifying a test substance but also for identifying, detecting, identifying, and searching for a test substance. For this reason, in the present invention, “measurement” is understood not only to quantitate the test substance but also to include the meaning of identification, detection, identification, and search of the test substance.

The liposome using a sugar-containing lipid thin film refers to a liposome produced by forming a thin film in a state where sugar is contained in the lipid constituting the liposome, and then adding a buffer solution to the thin film and gently vortexing. Furthermore, it is preferable to perform a treatment such as centrifugation or membrane filtration in order to remove those smaller than a predetermined size. This liposome was developed by those including the present inventors. Specifically, it can be produced by the method described in Non-Patent Document 3. The sugar contained in the liposome containing a sugar-containing lipid thin film is preferably at least one selected from the group consisting of fructose, mannose, glucose, and galactose.
On the surface of the liposome containing a sugar-containing lipid thin film, an antibody or a test substance is immobilized by a covalent bond. In the conventional ELISA method, an antibody or the like is immobilized by non-covalent bonding, and thus may be detached during the operation. On the other hand, in the present invention, since it is more strongly immobilized by a covalent bond, the possibility that an antibody or a test substance is detached is reduced, and precise measurement can be performed.
In order to covalently bond the test substance or antibody to the liposome, a cross-coupling reagent that binds to an amino group, thiol group, sugar chain, NHS ester, or the like can be used. As such a reagent, a commercially available one (for example, one commercially available as a crosslinker (http://www.funakoshi.co.jp/shiyaku/entry/3577.php)) can be used.
Moreover, in this invention, it is preferable that the solution used for a coupling | bonding process and a washing | cleaning process is a thing which does not contain surfactant. As such a solution, PBS (Phosphate Buffered Saline) can be used. PBS can be said to be essential in biochemical experiments, and since it is a solution that is always available, it is not necessary to use a special reagent. Further, if a solution containing a surfactant is used in any step, it is very difficult to completely remove the surfactant, and artifacts due to carry-over may occur. Furthermore, in a test system using liposomes that are lipid membranes, it is easier to obtain stable test results when as little surfactant as possible is used. In the conventional ELISA method, a solution containing a surfactant is commonly used (for example, PBST (Phosphate Buffered Saline with Tween or Triton)), and the influence of carry-over of the surfactant must always be considered. For these reasons, it can be said that the test system of the present invention which does not use a surfactant is very useful.

  In addition, the test substance is preferably any substance that can bind to the liposome including a low molecular substance, a high molecular substance, a virus particle, and a fungus body. Specific examples of the test substance or test substance recognized by the antibody bound to the liposome include the following. Cytokines (eg, interleukins (2, 3, 6, 10, etc.), interferons (α, β, γ), tumor necrosis factor (TNF), lymphotoxin c-kit, c-fms, EGF, PDGF, VEGF, TGF- β, BMP, activin, GM-CSF, LIF, NGF, Fas, CD40, IL-8, etc., apoptosis-related factors (eg, Fas, p53, etc.), transcriptional regulators (eg, Pax6, Pax8, etc.), tumor markers (For example, CEA (colon cancer, gastric cancer, pancreatic cancer marker), CA19-9 (pancreatic cancer, gallbladder cancer, bile duct cancer, ovarian cancer, endometrial cancer marker), AFP (hepatocellular carcinoma marker), CA125 (ovarian cancer marker) Etc.), neurotransmitters (acetylcholine, adrenaline (epinephrine), noradrenaline (norepinephrine), dopamine, serotonin, glycine, GABA, etc.), hormones (pituitary hormone releasing hormone, pituitary hormone release inhibitory hormone) , Oxytocin, vasopressin, corticotropin, corticotropin releasing hormone (factor) (CRH, CRF), melanocyte stimulating hormone, thyroid stimulating hormone (TSH), gonadotropin, growth hormone, prolactin, selectin, gastrin, Cholecystokinin, insulin, glucagon, leptin, calcitonin, parathyroid hormone, atrial natriuretic peptide, adrenaline (epinephrine), noradrenaline (norepinephrine), testosterone, estradiol, thyroxine, etc., virus (HIV (type 1, 2)) , Hepatitis virus (A, B, C, D, E type), influenza virus (A, B type), vesicular stomatitis virus, Semliki Forest virus, houl plaque virus, etc., bacteria (Helicobacter pylori, Legionlla pneumophila, Mycop lasma pneumoniae, Chlamiadia pneumoniae, etc.), plasma lipoprotein (high density lipoprotein (HDL), low density lipoprotein (LDL), etc.) and the like. Examples of infection protection and immune systems include fMet-Ler-Phe, complement, and antigen. Examples of transport proteins include LDL, HDL, transferrin, transcobalamin, egg yolk protein, macroglobulin, IgG, and IgA. For glycoproteins, Gal, Man / GlcNAc, GlcNAc, Man-6-P, etc., for plant lectins, concanavalin A, PHA, ricin, etc., for toxins, diphtheria toxin, cholera toxin, Examples include endotoxin. Further, as agonist (A) and antagonist (AG), succinylcholine (A), nicotine (A), d-tubocurarine (AG), gallamine (AG), hexamethonium (AG), α-bungarotoxin (AG), Carbachol (A), pilocarbine (A), carbachol (A), pirenzepine (AG), atropine (AG), scopolamine (AG), phenylephrine (A), clonidine (A), prazosin (AG), phenoxybenzamine (AG) ), Yohimbine (AG), isoproterenol (A), dobutamine (A), salbutamol (A), propranolol (AG), practolol (AG), butoxamine (AG), apomorphine (A), SKF38393 (A) , Apomorphine (A), Lislide (A), SCH23390 (AG), cis-Flupentixol (AG), Sulpiride (AG), Spiperone (AG), Haloperidol (AG), Muscimol (A), Baclofen (A) ), Bicuculline (AG), strychnine (AG), 2- Tilhistamine (A), 4-methylhistamine (A), isopromidine (A), mepyramine (AG), difendamine (AG), methiamide (AG), cimetidine (AG), methyl serzide (AG), LSD (AG ), Ketanserin (AG), morphine (A), ketocyclazocine (A), dynorphin (A), [D-Ara2, D-Leu5] enkephalin (A), β-endorphin (A), naloxone (AG), Mr2266 (AG), ICI154129 (AG) and the like.

  In addition to these, substances currently manufactured and sold as ELISA methods such as Mesothelin, Cardiotrophin-1, cardiovascular-related factors, prostaglandins, sRANKL, 2-Methoxyestradiol, MMP, immunoglobulin (IgM , IgG, IgA, IgD, IgE), Xenin25, obesity-related factors (adiponectin, leptin, resistin), bioactive peptides (Copeptin, Maxadilan), GFAP, MIC-1, GSTA, heart disease-related factors (morphine to LVV, angiotensin) , Aldosterone, bradykinin), Osteoprotegrin, plasminogen activator (PAI-1, uPA, tPA), chromogranin, adiponectin, CRP, βIG-H3, Podocalyxin, amyloid A, angiotensin, RIG-1, bradykinin, TL1A, Omentin1 , MxA, Calprotectin, NF-kB, protein A, insulin, Hsc70, Hsp90, cAMP, Sclerostin, allergens (Birch pollen allergen, Cat al lergen, Cockroach allergen, Dog allergen, Equine allergen, Fugi allergen, Mite allergen, Mouse allergen, Rat allergen, Short ragweed pollen allergen, Timothy pollen allergen, Peanut allergen), Hsp / anti-Hsp antibody, β-Amyloid, myoglobin, anti-pylori Bacteria antibody, heparanase, phosphorylated protein, steroid hormone, protein kinase inhibitor, hyaluronic acid, prostate secretory protein, GHRF, soluble CD147, NPY, apoptosis-related factor (Fas, CD178, Cytochrome c, p53, Perforin, TRAIL, TRAIL R1 , TRAIL R2, TRAIL R3, TRAIL R4, CD25, CD141, CD31, CD44, CD54, CD106, sCD138, CD95, CD178), natural toxin (Cylindrosper mopsin, Okadaic acid, TTX), albumin, cell growth factor (EGF, FGF) , IGF, TGF, NGF, BDNF, VEGF, G-CSF, GM-CSF, PDGF, EPO, TPO, HGF), prion protein, blood-related factors (α1-acid glycoprotein, α2-HS-glycoprotein, Antithrombin, Apolip oprotein, BNP, Factor X, Ferritin), environmental hormones (bisphenol A, alkylphenols, phthalates, benzopyrenes), agricultural chemicals (diuron, isoproturon, pentachlorophenol, dichlorophenoxyacetic acid, atrazine, triazine, alachlor, etc.), antibiotics (Sulfamesadine, chloramphenicol, tetracycline, streptomycin, AOZ, gentamicin, triclosan, etc.) are exemplified.

“Liposome” means a closed vesicle containing a lipid bilayer containing phospholipid (PL) and having an aqueous phase inside. Liposomes are divided into multilamellar vesicles (MLV) in which lipid bilayers are stacked in two or more layers, and liposomes with a single lipid bilayer (UV, unilamellar vesicle). It is done. UV is classified into small unilamellar vesicle (SUV), large unilamellar vesicle (LUV) and large liposome (giant liposome or giant vesicle (GV)) depending on the particle size. . In addition, giant liposomes include one having a lipid bilayer (GUV, giant unilamellar vesicel) and one having two or more lipid bilayers (GMV, giant multilamellar vesicle). GV is preferably used as the liposome used in the present invention (liposome utilizing saccharide-containing lipid thin film). The use of GV is preferable because when the test substance-bound liposome or antibody-bound liposome is prepared, the test substance or antibody is favorably disposed throughout the liposome, so that there is less variation in subsequent data. In addition, it is preferable to use GV because it can be precipitated by low-speed centrifugation.
In the present invention, a liposome containing a sugar-containing lipid thin film is used. When a thin film that is the basis of the liposome is produced, a GUV that has been difficult to produce stably in the past can be stably produced by incorporating a sugar (Non-patent Document 3). GMV can also be manufactured in large quantities.

Phospholipid means a substance containing phosphoric acid and lipid. Depending on the component, it is classified into a glycerophospholipid having a glycerol skeleton and a sphingophospholipid having a sphingosine skeleton. Examples of glycerophospholipid include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol. (PG), diphosphatidylglycerol (cardiolipin), phosphatidic acid (PA), etc. can be illustrated. Examples of sphingophospholipids include sphingomyelin. As the liposome used in the present invention, the above-mentioned various phospholipid components mixed at an arbitrary ratio can be used. For example, DOPC and DOPG can be the main components. However, PE is essential for binding proteins and the like to liposomes.
As a container for providing a liquid phase for reaction, a tube (including a plastic tube or a glass tube), a plate (a microtiter plate having 6 holes, 48 holes, 96 holes, or 384 holes) can be used. Examples of the detection method include radioimmunoassay, radioreceptor assay, enzyme immunoassay, fluorescence immunoassay, and chemiluminescence assay.

In the present invention, the inner wall surface of the first container and / or the second container is preferably previously subjected to a blocking treatment with a blocking agent that prevents nonspecific adsorption. In the conventional ELISA method, the antigen or antibody is adsorbed onto the wall surface and solidified, so that the adsorption of the antigen or antibody is inhibited if the blocking treatment is performed first. In contrast, in the LELIA method of the present invention, the entire inner wall surface of the container can be blocked in advance because the reaction is performed using liposomes floating in the liquid phase. For this reason, background data can be reduced sufficiently.
As a blocking agent for reducing nonspecific adsorption by blocking the inner wall surface of the container, a commercially available product for biochemical research can be used. Use of a blocking agent is preferable because specific binding of a substance that binds to an antigen or antibody becomes clearer.
The washing step includes a step of accumulating liposomes on the bottom and wall of the container and a step of adding and suspending the accumulated liposomes in an aqueous solution. Examples of the steps of accumulation include centrifugal accumulation, magnetic substance-encapsulated liposomes, and the like. A magnetic integration method using a magnet is exemplified.
The labeling enzyme used for the enzyme-labeled secondary antibody is not particularly limited. For example, enzymes such as alkaline phosphatase, peroxidase, β-galactosidase, and luciferase can be used. In the case of fluorescence immunoassay in which an enzyme-labeled secondary antibody is detected by fluorescence, for example, a secondary antibody labeled with a fluorescent substance such as Cy3, Cy5, fluorescein (FITC, etc.) can be used.

  According to the present invention, by using a liposome containing a sugar-containing lipid thin film in which an antibody or a test substance is bound to the membrane surface, the baseline is sufficiently reduced by reducing non-specific reaction, and detection is not conventionally performed. It is possible to provide LELIA (Liposome-based Enzyme-Linked ImmunoAssay), which is an enzyme immunoassay technology that specifically measures trace substances that have been possible. Further, according to the present invention, it is not necessary to add a surfactant to the solution in which the reaction is performed.

It is a figure which shows the image of the measuring method (LELIA method) in this invention. In this figure, the step of measuring the primary antibody 5 that reacts with the test substance 3 is shown. (A)-(R) is the schematic diagram which has shown in order that a process progresses and shows the mode in the tube 1, 1 ', 1' 'in each process. It should be noted that operations (M) to (N), which are operations between the steps indicated by the bent arrows on the upper side of the tube 1, are changed from the tube 1 to another tube 1 ′, and (Q) to (R) are the tube 1 ′. The operation of transferring the solution from 1 to another tube 1 ″ is shown. The operation between other processes is performed using the same tube. (A) The figure which shows a mode that the test substance coupling | bonding liposome which made the test substance 3 covalently bond to the surface of the liposome 2 was added to the tube 1 (1st container), (B) The tube 1 was centrifuged, and a test substance coupling | bonding liposome (C) The figure which shows a mode that the test substance coupling | bonding liposome which was precipitated and the test substance binding liposome which does not precipitate was removed, (C) The figure which shows a mode that the test substance coupling | bonding liposome was floated, (D) The tube 1 is distant again. Fig. 5 shows a state in which test substance-bound liposomes are precipitated and test substance-bound liposomes that are not precipitated are completely removed; (E) a sample containing primary antibody 5 that binds to test substance 3 and a buffer solution are added; The figure which shows a mode that the test substance binding liposome was suspended, (F) The tube 1 was centrifuged, the test substance and the primary antibody binding liposome were precipitated, and the unbound primary antibody 5 was removed. The figure which shows a child, (G) The figure which shows a mode that the buffer solution was added and the test substance and the primary antibody binding liposome were suspended, (H) The tube 1 was centrifuged again, and the test substance and the primary antibody binding liposome were The figure which shows a mode that it precipitated, (I) The figure which shows a mode that the enzyme-labeled secondary antibody 6 which recognizes the primary antibody 5, and the buffer solution were added, and the test substance and the primary antibody binding liposome were suspended, (J ) The tube 1 is centrifuged, the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome is precipitated, and the unbound enzyme-labeled secondary antibody is removed, (K) buffer is added, The figure which shows a mode that the test substance, the primary antibody, and the enzyme label | marker secondary antibody binding liposome were floated, (L) The tube 1 was centrifuged again and the test substance, the primary antibody, and the enzyme label secondary antibody binding liposome were precipitated. Figure showing the situation, (M The figure which shows a mode that the buffer solution was added and the test substance, the primary antibody, and the enzyme-labeled secondary antibody binding liposome were suspended, and the liquid of (N) and (M) was put into another tube 1 ′ (second container). The figure which shows a mode that it transferred, (O) The figure which shows a mode that the tube 1 'was centrifuged and the test substance, the primary antibody, and the enzyme-labeled secondary antibody binding liposome were precipitated, (P) of the enzyme-labeled secondary antibody 6 The figure which shows a mode that the substrate 7 (reactant 8) which reacts with an enzyme is added, the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome is suspended and reacted, (Q) The tube 1 ′ is centrifuged, It is a figure which shows a mode that a test substance, a primary antibody, and an enzyme label | marker secondary antibody binding liposome are precipitated, (R) It is a figure which shows a mode that the reaction material 8 in tube 1 'is isolate | separated and detected. It is a figure which shows the image of the measuring method (LELIA method) in this invention. In this figure, the process of measuring the test substance 3 that binds to the antibody 4 is shown. (A)-(U) has shown in order in which a process progresses, and is a schematic diagram which shows the mode in the tubes 1 and 1 'in each process. Note that (Q) to (R), which are operations between steps indicated by an arrow bent above the tube 1, indicate operations for transferring the solution from the tube 1 to another tube 1 '. The operation between other processes is performed using the same tube. (A) The figure which shows a mode that the antibody binding liposome which covalently bound the antibody 4 which recognizes the test substance 3 to the surface of the liposome 2 was added in the tube 1 (1st container), (B) The tube 1 was centrifuged. The figure which shows a mode that liposome 2 was settled and the liposome which does not settle was removed, (C) The figure which shows a mode that antibody-bound liposome was suspended by adding a buffer solution, (D) The tube 1 was centrifuged again. The figure which shows a mode that antibody-bound liposome was precipitated and the liposome which does not precipitate was removed completely, (E) The test substance 3 couple | bonded with the antibody 4 and a buffer solution were added, and the mode which floated the liposome 2 is shown. Figure, (F) The tube 1 is centrifuged to precipitate antibody / test substance binding liposomes, and the unbound test substance 3 is removed. (G) The buffer solution is added to bind the antibody / test substance. The figure which shows the state which floated the posome, (H) The figure which shows the mode which centrifuged the tube 1 again and precipitated the antibody and test substance binding liposome, (I) It reacts with the test substance 3 couple | bonded with the antibody 4 The figure which shows a mode that the antibody, the test substance, and the primary antibody binding liposome were suspended by adding the primary antibody 5 and the buffer, (J) The tube 1 was centrifuged, and the antibody, the test substance, and the primary antibody binding liposome were precipitated. (K) The figure which shows a mode that unbound primary antibody 5 was removed, (K) The figure which shows a mode that the buffer solution was added and the antibody, the test substance, and the primary antibody binding liposome were suspended, (L) Tube again 1 is a view showing a state in which an antibody / test substance / primary antibody-bound liposome is precipitated by centrifuging 1; (M) an enzyme-labeled secondary antibody 6 recognizing primary antibody 5 and a buffer solution are added to the antibody / test Substance, primary antibody, enzyme (N) The tube 1 is centrifuged to precipitate the antibody / test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome, and unbound enzyme-labeled secondary The figure which shows a mode that the antibody 6 was removed, (O) The figure which shows a mode that the buffer solution was added and the antibody, the test substance, the primary antibody, and the enzyme labeled secondary antibody liposome were suspended, (P) The tube 1 again. Is a diagram showing a state in which antibody, test substance, primary antibody, enzyme-labeled secondary antibody liposomes are precipitated by centrifuging, and (Q) a buffer solution is added to the antibody / test substance / primary antibody / enzyme-labeled secondary antibody The figure which shows a mode that the liposome was suspended, the figure which shows the mode which transferred the liquid of (R) (Q) to another tube 1 ', (S) The tube 1' was centrifuged, and an antibody, a test substance, and a primary antibody・ Precipitating enzyme-labeled secondary antibody liposomes (T) Substrate 7 (reactant 8) that reacts with the enzyme of the enzyme-labeled secondary antibody 6 is added to float the antibody / test substance / primary antibody / enzyme-labeled secondary antibody liposome. The figure which shows a mode of making it react, (U) It is a figure which shows a mode that the reaction substance 8 in tube 1 'is isolate | separated by precipitating an antibody, a test substance, a primary antibody, and an enzyme labeled secondary antibody liposome, and detecting it. . It is a graph which shows the test result which evaluated whether the test system was influenced by the presence or absence of surfactant. It is a graph which shows the test result which evaluated whether the test system was influenced by the presence or absence of blocking. It is a graph which shows the test result which evaluated whether the test system was influenced by the presence or absence of the container exchange before an enzyme reaction. It is a graph which shows the test result which evaluated whether the presence or absence of surfactant had influence on a test system by the presence or absence of the container exchange before an enzyme reaction. Whether the test substance hCRP can be detected specifically and in a concentration-dependent manner when the concentration of the primary antibody anti-hCRP antibody (Goat anti-hCRP) is changed by LELIA using hCRP-conjugated sGV It is a graph which shows the test result which evaluated this. We evaluated whether hCRP as a test substance could be detected specifically and in a concentration-dependent manner when the concentration of an enzyme-labeled secondary antibody (anti-IgG) was changed by LELIA using hCRP-conjugated sGV It is a graph which shows a test result. When LELIA using anti-hCRP antibody (anti-hCRP) -conjugated sGV was used, the enzyme-labeled secondary antibody dilution ratio was reduced to 1/10000, and the concentration of hCRP as the test substance was changed. It is a graph which shows the test result which evaluated whether it can detect dependently. When the concentration of enzyme-labeled secondary antibody (anti-IgG) is changed by LELIA using anti-hCRP antibody (anti-hCRP) -conjugated sGV, the test substance hCRP is detected specifically and concentration-dependently It is a graph which shows the test result which evaluated whether it was possible. When the enzyme-labeled secondary antibody dilution ratio was reduced to 1/5000 by LELIA using anti-hCRP antibody (anti-hCRP) -conjugated sGV and the concentration of hCRP as the test substance was changed, It is a graph which shows the test result which evaluated whether it can detect dependently. LELIA using human insulin-binding sGV can detect human insulin as a test substance specifically and in a concentration-dependent manner when the concentration of anti-human insulin antibody (Rabbit anti-insulin) is changed. It is a graph which shows the test result which evaluated whether or not. Whether the test substance human insulin can be detected specifically and concentration-dependently when the concentration of the enzyme-labeled secondary antibody (anti-IgG) is changed by LELIA using human insulin-binding sGV It is a graph which shows the test result evaluated. Test results of evaluating whether human insulin can be detected specifically and in a concentration-dependent manner when the concentration of human insulin, the test substance, is changed by LELIA using anti-human insulin monoclonal antibody-conjugated sGV It is a graph to show. Test substance when the dilution ratio of primary antibody anti-insulin antibody (Rabbit anti-inulin) was changed to 1/1000 by dilution with enzyme-labeled secondary antibody by LELIA using anti-human insulin monoclonal antibody-conjugated sGV It is a graph which shows the test result which evaluated whether human insulin which is this can be detected specifically and concentration-dependently. Can LELIA using anti-human insulin monoclonal antibody-conjugated sGV detect human insulin as a test substance specifically and in a concentration-dependent manner when the concentration of an enzyme-labeled secondary antibody (anti-IgG) is changed? It is a graph which shows the test result which evaluated whether or not. When the enzyme-labeled secondary antibody dilution ratio was set to 1/5000 by LELIA using Biotin-conjugated sGV, and the concentration of anti-Biotin antibody (Goat anti-Biotin) as the primary antibody was changed from 1 nM to 1 pM, It is a graph which shows the test result which evaluated whether Biotin which is a test substance can be detected specifically and concentration-dependently. We evaluated whether biotin as a test substance could be detected specifically and concentration-dependently when the concentration of enzyme-labeled secondary antibody (anti-IgG) was changed by LELIA using Biotin-conjugated sGV It is a graph which shows a test result. When LELIA using Biotin-conjugated sGV was used, the enzyme-labeled secondary antibody dilution ratio was 1/1000, and the concentration of the primary antibody anti-Biotin antibody (Goat anti-Biotin) was changed from 10 pM to 0.1 pM. It is a graph which shows the test result which evaluated whether Biotin which is a test substance can be detected specifically and concentration-dependently. Test that evaluates whether Biotin can be detected specifically and in a concentration-dependent manner when the concentration of Biotin, a test substance, is changed by LELIA using anti-Biotin antibody (Rabbit anti-Biotin) binding sGV It is a graph which shows a result. When the concentration of anti-biotin antibody (Goat anti-Biotin) is changed by LELIA using anti-Biotin antibody (Rabbit anti-Biotin) binding sGV, the concentration of Biotin, which is the test substance, is increased specifically. It is a graph which shows the test result which evaluated whether it can detect dependently. When the concentration of the enzyme-labeled secondary antibody (anti-IgG) was changed by LELIA using anti-Biotin antibody (Rabbit anti-Biotin) -conjugated sGV, the test substance Biotin was specifically and concentration-dependent. It is a graph which shows the test result which evaluated whether it can detect.

Embodiments of the present invention will be described in detail with reference to the drawings. The technical scope of the present invention is not limited by the following embodiments, and can be implemented with various modifications without changing the gist thereof. The technical scope of the present invention extends to an equivalent range.
FIG. 1 shows an image at the time of LELIA measurement. In FIG. 1, the description of the liquid phase (such as a buffer solution during the reaction) is omitted for the sake of simplicity (the same applies to FIG. 2).
FIGS. 1A and 1B are diagrams (A) showing how test substance-bound liposomes (covalent binding step) in which test substance 3 is covalently bonded to the surface of liposome 2 are added to tube 1 (first container). Steps), (B) The tube 1 is centrifuged to precipitate test substance-bound liposomes, and the test substance-bound liposomes that do not precipitate are removed. (C) A buffer is added to float the test substance-bound liposomes. (D) The figure which shows a mode that the tube 1 was centrifuged again and the test substance binding | bonding liposome was precipitated ((B)-(D) corresponds to the "liposome washing | cleaning process" in this invention. ), (E) A diagram (primary antibody binding step) showing a state in which a test substance-bound liposome is suspended by adding a sample containing a primary antibody 5 that binds to the test substance 3 and a buffer solution, and (F) tube 1 The Fig. 5 shows a state in which test substance / primary antibody-bound liposomes are precipitated and unbound primary antibody 5 is removed, (G) buffer is added, and test substance / primary antibody-bound liposomes are suspended. The figure which shows the mode (H) which again centrifuged the tube 1 and precipitated the test substance and the primary antibody binding liposome ((F)-(H) correspond to the "primary antibody washing process" in this invention) ), (I) The figure which shows the state which added the enzyme labeled secondary antibody 6 and the buffer solution which recognize the primary antibody 5, and the buffer solution, and suspended the test substance and the primary antibody binding liposome (second antibody binding process), ( J) The tube 1 is centrifuged, the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome is precipitated, and the unbound enzyme-labeled secondary antibody is removed. (K) A buffer solution is added. , Test substance, primary antibody, enzyme (L) The tube 1 is centrifuged again, and the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome is precipitated ((J) )-(L) corresponds to “secondary antibody washing step” in the present invention), and (M) a buffer solution is added to suspend the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome. The figure which shows the state (transfer process) which transferred the liquid of (N) and (M) to another tube 1 '(2nd container), (O) The tube 1' is centrifuged, a test substance and primary The figure which shows a mode that the antibody-enzyme labeled secondary antibody binding liposome was precipitated, (P) Substrate 7 (reactant 8) which reacts with the enzyme of the enzyme labeled secondary antibody 6 was added, and the test substance / primary antibody / Enzyme-labeled secondary antibody-bound liposomes are suspended and reacted (Q) The figure which shows a mode that the tube 1 'was centrifuged and the test substance, the primary antibody, and the enzyme labeled secondary antibody binding liposome were precipitated. (R) The reaction product 8 in the tube 1' was separated. It is a figure (detection process) which shows a mode that it detects.
By this LELIA, the test substance 3 can be measured (including identification, detection, identification, search, etc.).

In FIG. 2, the image at the time of the measurement of LELIA by another scheme was shown. FIG. 2 shows a state in which (A) an antibody-binding liposome (covalent binding step) in which an antibody 4 that recognizes the test substance 3 is covalently bonded to the surface of the liposome 2 is added to the tube 1 (first container). Figure showing (container adsorption process), (B) Centrifugation of tube 1 to precipitate liposome 2, and figure showing removal of non-precipitated liposome, (C) Add buffer to float antibody-bound liposome (D) The figure which shows a mode that the tube 1 was centrifuged again and the antibody binding liposome was precipitated ((B)-(D) corresponds to the "liposome washing process" in this invention), (E) The figure which shows the state which added the test substance 3 and buffer solution couple | bonded with the antibody 4, and suspended the liposome 2 (test substance binding process), (F) The tube 1 was centrifuged, and an antibody and a test substance Bound liposomes The figure which shows a mode that it was made to stir and the unbound test substance 3 was removed, (G) The figure which shows a mode that the buffer solution was added and the antibody and test substance coupling | bonding liposome were suspended, (H) The tube 1 is again shown. The figure which shows a mode that the antibody and test substance binding | bond | coupling liposome were precipitated by centrifuging ((F)-(H) corresponds to the "test substance washing | cleaning process" in this invention), (I) The test substance couple | bonded with the antibody 4 3 shows a state in which the antibody / test substance / primary antibody-binding liposome is suspended by adding the primary antibody 5 and the buffer solution reacting to 3 (primary antibody binding step), (J) the tube 1 is centrifuged, and the antibody -Test substance / primary antibody-bound liposomes are precipitated and unbound primary antibody 5 is removed. (K) Buffer is added to float antibody / test substance / primary antibody-bound liposomes. (L) Again, The figure which shows a mode that the antibody 1, the to-be-tested substance, and the primary antibody binding liposome were precipitated by centrifuging (1) (the (J)-(L) is the "primary antibody washing process" in this invention)), (M) primary antibody 5 The figure which shows a mode that the enzyme-labeled secondary antibody 6 which recognizes and buffer solution were added, and the antibody, the test substance, the primary antibody, and the enzyme-labeled secondary antibody-bound liposome were suspended (secondary antibody binding step), N) The tube 1 is centrifuged to precipitate the antibody / test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome, and the unbound enzyme-labeled secondary antibody 6 is removed. (O) Buffer solution The figure which shows a mode that the antibody, the test substance, the primary antibody, and the enzyme-labeled secondary antibody liposome were added and suspended, (P) The tube 1 was centrifuged again, and the antibody, the test substance, the primary antibody, and the enzyme-labeled secondary Figure showing how antibody liposomes are precipitated ( (N)-(P) corresponds to the “secondary antibody washing step” in the present invention), (Q) a buffer solution is added to float the antibody / test substance / primary antibody / enzyme-labeled secondary antibody liposome. The figure which shows the mode, (R) The figure which shows the state which transferred the liquid of (Q) to another tube 1 '(transfer process), (S) The tube 1' is centrifuged, and an antibody, a test substance, and a primary antibody -Diagram showing how enzyme-labeled secondary antibody liposomes are precipitated, (T) Substrate 7 (reactant 8) that reacts with enzyme of enzyme-labeled secondary antibody 6 is added, and antibody, test substance, primary antibody, The figure which shows an enzyme-labeled secondary antibody liposome floating and reacting, (U) Precipitating antibody / test substance / primary antibody / enzyme-labeled secondary antibody liposome, and separating / detecting the reactant 8 in the tube 1 ′ It is a figure (detection process) which shows a mode that it does. This LELIA can also measure the test substance 3 (including identification, detection, identification, search, etc.).
Next, the present embodiment will be described in more detail by describing an example in which LELIA shown in FIGS. 1 and 2 is embodied. As test substances, biotin having a molecular weight of about 240, human insulin having a molecular weight of about 5,800, and human C-reactive protein having a molecular weight of about 130,000 were used.
<Experimental materials and methods>

1. Materials Phospholipids for preparing liposomes are 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (NOF Corporation), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine ( DOPS) (NOF Corporation) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (NOF Corporation) were used.
Liposome membrane binding antigens are Human C-Reactive Protein (hCRP) (Life Diagnostics, Inc.), Insulin human (Roche Ltd.) or 6- (Biotinylamino) hexanonic acid N-hydroxysuccinimide ester (Biotin-AC ₅ -OSu) (DOJINDO LABORATORIES) was used.
Liposome membrane binding antibodies include Rabbit IgG to Human C-Reactive Protein (Rabbit anti-hCRP) (Good Biotech Corp.), anti-human insulin monoclonal antibody (BALB / C mouse) (Nippon Biotest Laboratories Inc.) or Anti-Biotin. In Rabbit, IgG fraction (Rabbit anti-Biotin) (Polysciences, Inc.) was used.
The ligands and antibodies used in LELIA are hCRP, Insulin human (Roche Ltd.), Biotin (Sigma-Aldrich Co.), Goat anti-Human CRP (Goat anti-hCRP) (Bethyl Laboratories, Inc.), Anti-Insulin. , Rabbit Polyclonal (ABBIOTEC, LLC), Goat anti-Biotin (Bethyl Laboratories, Inc.), Anti-Rabbit IgG (H + L chain) -HRP (Medical & Biological Laboratories Co., Ltd.) and Anti-Goat IgG- HRP (Medical & Biological Laboratories Co., Ltd.) was used.

2. Preparation of liposome A relatively small sugar-containing lipid thin film-based giant liposome (sGV) was prepared as follows. Remove the mixed solution of phospholipid dissolved in chloroform and fructose dissolved in methanol (DOPC / DOPS / DOPE / Fructose = 1/5/4/10, volume ratio of chloroform to methanol 2: 1) by vacuum vortexing. After forming the lipid film, reduce the pressure for another 1 hour, add 1 ml of 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer to the phospholipid in the form of a thin film, gently vortex, Prepared. Next, the prepared liposomes were subjected to pressure filtration with a 10 μm polycarbonate membrane filter to obtain sGV. In order to remove liposomes other than sGV, centrifugation (19,500 × g, 20 min, 4 ° C.) was performed, and the supernatant was removed, and the resulting precipitate was buffered with 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5). It was suspended in the liquid and centrifuged again (19,500 × g, 20 min, 4 ° C.). The same operation was repeated 4 times to prepare sGV as uniform as possible. The prepared sGV was filled with argon gas and stored at 4 ° C.

3. Determination of liposome concentration The liposome concentration was expressed in terms of phospholipid concentration. The phospholipid concentration was obtained by wet-decomposing phospholipids with hydrogen peroxide and sulfuric acid and coloring the inorganic phosphorus in the resulting decomposition solution with Fiske-Subbarow reagent. First, 4 mmol of H ₂ SO ₄ was added to the KH ₂ PO ₄ solution used as a sample and a control, heated at 170 ° C. for 30 minutes or more, and after air cooling, hydrogen peroxide was added to 6%, at 170 ° C. Heated for 30 minutes. Next, 4.6 ml of 0.25NH ₂ SO ₄ /1.8 mM (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O solution was added to the air-cooled sample and control, vortexed, and a color reagent (10 mM 1-Amino) was added. After adding 0.2 ml of -2-naphthol-4-sulfonic acid / 4 mM Na ₂ SO ₃ /15% Sodium Hydrogensulfite) and vortexing, the mixture was heated in boiling water for 10 minutes. The absorbance of the air-cooled sample and KH ₂ PO ₄ used as a control was measured at 830 nm to determine the phosphorus content in the sample.
4). Determination of protein concentration In the production of antigen and antibody membrane-bound liposomes, the protein concentration was determined by protein quantification by the Bradford method. After adjusting the total volume to 1.3 ml with 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) in any amount of protein solution and 2 mg / ml BSA 2, 4, 6, 8 μl used as a control, Bio- 0.2 ml of Rad Protein Assay (Bio-Rad Laboratories, Inc.) was added and vortexed. After standing at room temperature for 5 minutes, the absorbance was measured at 595 nm to determine the concentration.

<LELIA and results>
I. Basic operating conditions (1) Buffer 1
Here, the effect of Triton X-100 on the non-specific reaction between CRP and sGV was examined. Although blocking was performed, the container was not replaced.
<Method> First, the sample tube was washed with PBST or PBS (pH 7.2) and then 0.5 ml of 3% Block Ace (DS Pharma Biomedical Co., Ltd) was added, followed by incubation at 37 ° C. for 1 hour for blocking. . After blocking, remove the blocking solution, wash with PBST or PBS, add 100 μl each of sGV or 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer, and centrifuge (19,500 × g, 20 min, 4 ° C.) It was. After centrifugation, the supernatant was removed, the precipitate was suspended in PBST or PBS 0.5 ml, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample. Next, the supernatant was removed, 50 μl of Goat anti-hCRP antibody solution diluted to an arbitrary concentration with PBST or PBS was added and suspended, and incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBST or PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBST or PBS is added to suspend the precipitate, and then centrifuged (19,500 × g, 20 min, 4 ° C.) to wash the sample tube. Furthermore, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBST or PBS was added and suspended, and the mixture was incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBST or PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBST or PBS is added to suspend the precipitate, and then centrifuged (19,500). Xg, 20 min, 4 ° C.) to wash the sample. After performing the same washing operation four times, the precipitate was suspended in PBST or PBS 0.5 ml, and centrifuged (19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color is developed and 50 μl of 2N H ₂ SO ₄ is added to stop the reaction. Then, centrifugation (19,500 × g, 20 min, 4 ° C.) is performed, and the above is performed using a microplate reader (MULTISKAN JX, Thermo Fisher Scientific, Inc.). The absorbance of Kiyoshi at 492 nm (A ₄₉₂ ) was measured.
<Results> hCRP sGV and buffer when the concentration of hCRP is 1 nM, the concentration of anti-hCRP antibody is 5 nM, and the dilution ratio of anti-IgG antibody, which is a secondary antibody against anti-hCRP antibody, is constant at 1: 10000. The difference ΔA in reactivity with the solution was 0.098 when the buffer solution was PBST and 0.076 when the buffer solution was PBS, and there was no significant difference depending on the presence or absence of Triton X-100 (FIG. 3).

(2) Blocking Here, the effect of blocking in the non-specific reaction between CRP and sGV was examined. Surfactant addition and container exchange were not performed.
<Method> First, the sample tube was washed with PBS (pH 7.2), and then the sample tube was blocked and the sample tube was not. When blocking was performed, 0.5 ml of 3% Block Ace (DS Pharma Biomedical Co., Ltd) was added and incubated at 37 ° C. for 1 hour. When blocking is performed, the blocking solution is removed and washed with PBS. 100 μl of sGV or 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer is added, and centrifuged (19,500 × g, 20 min, 4 ° C.). ) After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample. Next, the supernatant was removed, 50 μl of Goat anti-hCRP antibody solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBS was added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS was added to suspend the precipitate and centrifuged again (19,500 × g, 20 min, 4 ° C) to wash the sample tube. Further, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) 4 ° C.) to wash the sample. After performing the same washing operation four times, the precipitate was suspended in 0.5 ml of PBS and centrifuged (19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color is developed and 50 μl of 2N H ₂ SO ₄ is added to stop the reaction. Then, centrifugation (19,500 × g, 20 min, 4 ° C.) is performed, and the above is performed using a microplate reader (MULTISKAN JX, Thermo Fisher Scientific, Inc.). The absorbance of Kiyoshi at 492 nm (A ₄₉₂ ) was measured.
<Results> hCRP sGV and buffer when the concentration of hCRP is 1 nM, the concentration of anti-hCRP antibody is 5 nM, and the dilution ratio of anti-IgG antibody, which is a secondary antibody against anti-hCRP antibody, is constant at 1: 10000. The difference ΔA in reactivity with the solution was 0.082 and 0.273, respectively, when blocking was performed and when blocking was not performed, and it was found that non-specific binding of hCRP to sGV was decreased when blocking. In addition, the value of the buffer solution alone was greatly reduced to 0.905 without blocking and 0.240 with blocking (FIG. 4).
From this result, it was found that blocking is essential.

(3) Container (transfer process)
Here, the effect of container exchange in the non-specific reaction between CRP and sGV was examined. Although blocking was performed, no surfactant was added.
<Method> First, the sample tube was washed with PBS (pH 7.2), 0.5 ml of 3% Block Ace (DS Pharma Biomedical Co., Ltd) was added, and the mixture was incubated at 37 ° C. for 1 hour for blocking. After blocking, the blocking solution was removed and washed with PBS. 100 μl each of sGV or 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer was added and centrifuged (19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample. Next, the supernatant was removed, 50 μl of Goat anti-hCRP antibody solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBS was added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS was added to suspend the precipitate and centrifuged again (19,500 × g, 20 min, 4 ° C) to wash the sample tube. Further, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) 4 ° C.) to wash the sample. After performing the same washing operation 4 times, suspend the precipitate in 0.5 ml of PBS, and transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour. The precipitate was suspended and centrifuged (19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color is developed and 50 μl of 2N H ₂ SO ₄ is added to stop the reaction. Then, centrifugation (19,500 × g, 20 min, 4 ° C.) is performed, and the above is performed using a microplate reader (MULTISKAN JX, Thermo Fisher Scientific, Inc.). The absorbance of Kiyoshi at 492 nm (A ₄₉₂ ) was measured.
<Results> hCRP sGV and buffer when the concentration of hCRP is 1 nM, the concentration of anti-hCRP antibody is 5 nM, and the dilution ratio of anti-IgG antibody, which is a secondary antibody against anti-hCRP antibody, is constant at 1: 10000. The difference ΔA in the reactivity with the liquid is 0.144 when there is no container replacement, 0.103 when there is a container replacement, and the value of the buffer solution is greatly reduced to 0.218 without container replacement and 0.040 with container replacement. (FIG. 5).
From this result, it was found that it is extremely effective to replace the container before the enzyme reaction and provide a transfer step.

(4) Buffer 2
Here, the effect of Triton X-100 on the nonspecific reaction between CRP and sGV when blocking and container exchange were performed was examined.
<Method> First, the sample tube was washed with PBST or PBS (pH 7.2) and then 0.5 ml of 3% Block Ace (DS Pharma Biomedical Co., Ltd) was added, followed by incubation at 37 ° C. for 1 hour for blocking. . After blocking, remove the blocking solution, wash with PBST or PBS, add 100 μl each of sGV or 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer, and centrifuge (19,500 × g, 20 min, 4 ° C.) It was. After centrifugation, the supernatant was removed, the precipitate was suspended in PBST or PBS 0.5 ml, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample. Next, the supernatant was removed, 50 μl of Goat anti-hCRP antibody solution diluted to an arbitrary concentration with PBST or PBS was added and suspended, and incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBST or PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBST or PBS is added to suspend the precipitate, and then centrifuged (19,500 × g, 20 min, 4 ° C.) to wash the sample tube. Furthermore, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBST or PBS was added and suspended, and the mixture was incubated at 37 ° C. for 1 hour. After the reaction, 0.5 ml of PBST or PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBST or PBS is added to suspend the precipitate, and then centrifuged (19,500). Xg, 20 min, 4 ° C.) to wash the sample. After performing the same washing operation 4 times, suspend the precipitate in PBST or PBS 0.5 ml, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour, and centrifuge (19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color is developed and 50 μl of 2N H ₂ SO ₄ is added to stop the reaction. Then, centrifugation (19,500 × g, 20 min, 4 ° C.) is performed, and the above is performed using a microplate reader (MULTISKAN JX, Thermo Fisher Scientific, Inc.). The absorbance of Kiyoshi at 492 nm (A ₄₉₂ ) was measured.

<Results> hCRP sGV and buffer when the concentration of hCRP is 1 nM, the concentration of anti-hCRP antibody is 5 nM, and the dilution ratio of anti-IgG antibody, which is a secondary antibody against anti-hCRP antibody, is constant at 1: 10000. The difference ΔA in reactivity with the solution was 0.097 when the buffer solution was PBST, and 0.103 when the buffer solution was PBS. Even when the container was replaced, there was no significant difference in the presence or absence of Triton X-100 (FIG. 6). ).
From the results of (1) and (4) above, it was found that a surfactant was unnecessary as a buffer solution. For this reason, in the present invention, PBS containing no surfactant may be used. PBS can be said to be essential in biochemical experiments, and since it is a solution that is always available, it is not necessary to use a special reagent. In addition, if a solution containing a surfactant is used in any step, it is very difficult to completely remove the surfactant. In the subsequent step, artifacts due to carry-over of the surfactant are caused. May occur. Furthermore, in a test system using liposomes that are lipid membranes, it is easier to obtain stable test results when as little surfactant as possible is used. In the conventional ELISA method, a solution containing a surfactant is commonly used (for example, PBST), and the influence of carry-over of the surfactant must always be considered. For these reasons, it can be said that the test system of the present invention which does not use a surfactant is very useful.

II. Membrane-bound sGV
(1) hCRP
<Method> Succinimidyl 4-formylbenzoate (SFB) (Thermo Fisher Scientific, Inc.) dissolved in Dimethylsulfoxide (DMSO) and sGV were mixed at a molar ratio of 2: 5 and reacted at 27 ° C. for 3 hours. SFB modified sGV was prepared. Further, succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) dissolved in DMSO and hCRP were mixed at a molar ratio of 20: 1 and reacted at 27 ° C. for 1 hour to prepare SANH-modified hCRP. The SFB-modified sGV and SANH-modified hCRP obtained as described above were used in 10 mM MES-NaOH / 100 mM NaCl (pH 4.7) buffer solution using Slide-A-Lyzer MINI Dialysis Units (Thermo Fisher Scientific, Inc.). And dialyzed overnight. After dialysis, the concentration of SFB-modified sGV and SANH-modified hCRP was determined, and SFB-modified sGV and SANH-modified hCRP were mixed at a molar ratio of 4000: 1 and reacted at 27 ° C for 3 hours, and then 1M Tris The solution was added to neutralize the pH to produce hCRP membrane-bound sGV (covalent binding step). Thereafter, SANH-modified hCRP, SFB-modified sGV and hCRP membrane-bound sGV were centrifuged (19,500 × g, 20 min, 4 ° C.), and the amount of protein in the supernatant was measured. Asked.
Binding rate (%) = 1-[(the amount of protein in the supernatant after centrifugation of hCRP membrane-bound sGV−the amount of protein in the supernatant after centrifugation of SFB-modified sGV) / (the protein in the supernatant after centrifugation of SANH-modified hCRP) Amount −10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) protein amount)] × 100
<Results> As a result of the above measurement, the binding rate of hCRP membrane-bound sGV was 98.5%.

(2) Rabbit anti-hCRP antibody <Method> First, Rabbit anti-hCRP antibody dissolved in 20 mM MES / 0.15M NaCl (pH 5.8) in order to increase the binding rate of Rabbit anti-hCRP antibody membrane-bound sGV sufficiently. The -hCRP antibody was dialyzed overnight in 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer using Slide-A-Lyzer MINI Dialysis Units to replace the solvent. Next, SFB and sGV dissolved in DMSO were mixed at a molar ratio of 9: 1 and reacted at 27 ° C. for 3 hours to prepare SFB-modified sGV. In addition, SANH dissolved in DMSO and Rabbit anti-hCRP antibody after dialysis were mixed at a molar ratio of 120: 1 and reacted at 27 ° C. for 1 hour to prepare SANH-modified Rabbit anti-hCRP antibody ( Covalent binding step). The SFB-modified sGV and SANH-modified Rabbit anti-hCRP antibodies thus obtained were dialyzed overnight in 10 mM MES-NaOH / 100 mM NaCl (pH 4.7) buffer using Slide-A-Lyzer MINI Dialysis Units. Went. After dialysis, the concentrations of SFB-modified sGV and SANH-modified Rabbit anti-hCRP antibody were determined, and SFB-modified sGV and SANH-modified Rabbit anti-hCRP antibody were mixed at a molar ratio of 4000: 1 at 27 ° C. After performing the reaction for 1 hour, 1M Tris solution was added to neutralize the pH, and Rabbit anti-hCRP antibody membrane-bound sGV was prepared. Then, SANH-modified Rabbit anti-hCRP antibody, SFB-modified sGV and Rabbit anti-hCRP antibody membrane-bound sGV were centrifuged (19,500 × g, 20 min, 4 ° C.), and the fluorescence intensity of the supernatant was measured. Was used to determine the binding rate of Rabbit anti-hCRP antibody membrane-bound sGV.
Binding rate (%) = 1-[(fluorescence intensity of supernatant after centrifugation of Rabbit anti-hCRP antibody membrane-bound sGV−fluorescence intensity of supernatant after centrifugation of SFB-modified sGV) / (SANH-modified Rabbit anti-hCRP antibody Fluorescence intensity of supernatant after centrifugation of -10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) fluorescence intensity)] × 100
<Results> As a result of the above measurement, the binding rate of Rabbit anti-hCRP antibody membrane-bound sGV was 88.7%.

(3) Human insulin <Method> Succinimidyl 4-formylbenzoate (SFB) (Thermo Fisher Scientific, Inc.) dissolved in dimethylsulfoxide (DMSO) and sGV are mixed at a molar ratio of 2: 5 and mixed at 27 ° C. Time reaction was performed to prepare SFB-modified sGV. Further, succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) dissolved in DMSO and human insulin were mixed at a molar ratio of 20: 1 and reacted at 27 ° C. for 1 hour to prepare SANH-modified human insulin. The SFB-modified sGV and SANH-modified human insulin thus obtained were added to a 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer solution using Slide-A-Lyzer MINI Dialysis Units (Thermo Fisher Scientific, Inc.). Dialysis was performed overnight. After dialysis, determine the concentration of SFB-modified sGV and SANH-modified human insulin, mix SFB-modified sGV and SANH-modified human insulin at a molar ratio of 4000: 1, mix at 27 ° C for 6 hours, and add at 4 ° C. After overnight reaction, human insulin membrane-bound sGV was prepared (covalent binding step). Thereafter, SANH-modified human insulin, SFB-modified sGV, and human insulin membrane-bound sGV were centrifuged (19,500 × g, 20 min, 4 ° C.), and the fluorescence intensity of the supernatant was measured. The binding rate of was determined. However, since human insulin has no tryptophan residue and the fluorescence of tyrosine residues is quenched by energy transfer to SANH, it was evaluated by the fluorescence intensity of SANH at 393 nm.
Binding rate (%) = 1-[(fluorescence intensity of supernatant after centrifugation of human insulin membrane-bound sGV−fluorescence intensity of supernatant after centrifugation of SFB-modified sGV) / (supernatant after centrifugation of SANH-modified human insulin) Fluorescence intensity-10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) fluorescence intensity)] x 100
<Results> As a result of the above measurement, the binding rate of human insulin membrane-bound sGV was 93.3%.

(4) Mouse Anti-Human Insulin Monoclonal Antibody <Method> First, in order to increase the binding rate of the prepared mouse anti-human insulin monoclonal antibody membrane-bound sGV, it is dissolved in 20 mM MES / 0.15M NaCl (pH 5.8). The mouse anti-human insulin monoclonal antibody was dialyzed overnight in 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer using Slide-A-Lyzer MINI Dialysis Units to replace the solvent. Next, SFB and sGV dissolved in DMSO were mixed at a molar ratio of 9: 1 and reacted at 27 ° C. for 3 hours to prepare SFB-modified sGV. In addition, SANH in DMSO and Mouse anti-human insulin monoclonal antibody after dialysis were mixed at a molar ratio of 120: 1 and reacted at 27 ° C for 1 hour to prepare SANH-modified Mouse anti-human insulin monoclonal antibody. (Covalent bonding step). The SFB-modified sGV and SANH-modified Mouse anti-human insulin monoclonal antibodies obtained in this manner were used overnight in 10 mM MES-NaOH / 100 mM NaCl (pH 4.7) buffer using Slide-A-Lyzer MINI Dialysis Units. Dialysis was performed. After dialysis, the concentration of SFB-modified sGV and SANH-modified Mouse anti-human insulin monoclonal antibody was determined, and SFB-modified sGV and SANH-modified Mouse anti-human insulin monoclonal antibody were mixed at a molar ratio of 4000: 1 to 27 ° C. After reacting for 3 hours, 1M Tris solution was added to neutralize the pH, and Mouse anti-human insulin monoclonal antibody membrane-bound sGV was prepared. Thereafter, SANH-modified Mouse anti-human insulin monoclonal antibody, SFB-modified sGV and Mouse anti-human insulin monoclonal antibody membrane-bound sGV were centrifuged (19,500 × g, 20 min, 4 ° C.), and the fluorescence intensity of the supernatant was measured. The binding rate of Mouse anti-human insulin monoclonal antibody membrane-bound sGV was determined by the following formula.
Binding rate (%) = 1-[(Mouse anti-human insulin monoclonal antibody membrane-bound sGV fluorescence intensity after centrifugation-SFB-modified sGV supernatant fluorescence intensity after centrifugation) / (SANH-modified Mouse anti-human insulin Fluorescence intensity of supernatant after centrifugation of monoclonal antibody-Fluorescence intensity of 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5))] x 100
<Results> As a result of the above measurement, the binding rate of Mouse anti-human insulin monoclonal antibody membrane-bound sGV was 96.4%.

(5) Biotin
<Method> Biotin-AC ₅ -Osu dissolved in DMSO and sGV were mixed at a molar ratio of 8: 3 and reacted at 27 ° C for 3 hours. Then, Slide-A-Lyzer MINI Dialysis Units (Thermo Fisher Scientific, Inc.) was dialyzed overnight in 10 mM MES-NaOH / 100 mM NaCl (pH 4.7) buffer. After dialysis, the sample is neutralized with 1M Tris solution, centrifuged (19,500 xg, 20 min, 4 ° C), the supernatant is removed, and the precipitate is precipitated with 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5). ) To produce Biotin membrane-bound sGV (covalent binding step). The produced Biotin membrane-bound sGV was directly subjected to LELIA.

(6) Rabbit anti-Biotin antibody <Method> SFB and sGV dissolved in DMSO were mixed at a molar ratio of 9: 1 and reacted at 27 ° C. for 3 hours to prepare SFB-modified sGV. In addition, SANH and Rabbit anti-Biotin antibody dissolved in DMSO were mixed at a molar ratio of 120: 1 and reacted at 27 ° C. for 1 hour to prepare SANH-modified Rabbit anti-Biotin antibody. The SFB-modified sGV and SANH-modified Rabbit anti-Biotin antibodies thus obtained were dialyzed overnight in 10 mM MES-NaOH / 100 mM NaCl (pH 4.7) buffer using Slide-A-Lyzer MINI Dialysis Units. Went. After dialysis, the concentration of SFB-modified sGV and SANH-modified Rabbit anti-Biotin antibody was determined, and SFB-modified sGV and SANH-modified Rabbit anti-Biotin antibody were mixed at a molar ratio of 4000: 1 and reacted at 27 ° C for 3 hours. Then, 1M Tris solution was added to neutralize the pH, and Rabbit anti-Biotin antibody membrane-bound sGV was prepared (covalent binding step). Thereafter, SANH-modified Rabbit anti-Biotin antibody, SFB-modified sGV and Rabbit anti-Biotin antibody membrane-bound sGV were centrifuged (19,500 × g, 20 min, 4 ° C.), and the amount of protein in the supernatant was measured. Was used to determine the binding rate of Rabbit anti-Biotin antibody membrane-bound sGV.
Binding rate (%) = 1-[(absorbance of supernatant after centrifugation of Rabbit anti-Biotin antibody membrane-bound sGV−absorbance of supernatant after centrifugation of SFB-modified sGV) / (centrifugation of SANH-modified Rabbit anti-Biotin antibody) Absorbance of the supernatant after -10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5))) x 100
<Results> As a result of the above measurement, the binding rate of Rabbit anti-Biotin antibody membrane-bound sGV was 94.9%.

III. Membrane-bound sGV-LELIA
(1) hCRP
The usefulness of LELIA in the specific reaction between hCRP membrane-bound sGV and Goat anti-hCRP was examined.
<Method> First, after washing the sample tube with PBS (pH 7.2), 0.5 ml of 3% Block Ace (DS Pharma Biomedical Co., Ltd.) was added and incubated at 37 ° C. for 1 hour for blocking (blocking). processing). After blocking, the blocking solution was removed and washed with PBS. The prepared hCRP membrane-bound sGV (hCRP amount-sGV amount = 1 μg-30 nmol / 100 μl) sample, SFB-modified sGV (sGV amount 30 nmol / 100 μl, control) or 10 mM HEPES- 100 μl of NaOH / 100 mM NaCl (pH 7.5) buffer was added, respectively, and centrifuged (19,500 × g, 20 min, 4 ° C.) (container adsorption step). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample (liposome washing step). Next, the supernatant was removed, 50 μl of Goat anti-hCRP antibody solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (primary antibody binding step). After the reaction, 0.5 ml of PBS was added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS was added to suspend the precipitate and centrifuged again (19,500 × g, 20 min, 4 ° C) to wash the sample tube (primary antibody washing step). Further, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (secondary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample (secondary antibody washing step). After performing the same washing operation four times, suspend the precipitate in 0.5 ml of PBS, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour (transfer step), and centrifuge ( 19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color is developed and 50 μl of 2N H ₂ SO ₄ is added to stop the reaction. Then, centrifugation (19,500 × g, 20 min, 4 ° C.) is performed, and the above is performed using a microplate reader (MULTISKAN JX, Thermo Fisher Scientific, Inc.). The absorbance (A ₄₉₂ ) of Kiyoshi at 492 nm was measured (detection step).

<Results> First, in the case of hCRP membrane-bound sGV when the dilution ratio of anti-IgG antibody to Goat anti-hCRP antibody is constant at 1: 10000 and the concentration of Goat anti-hCRP antibody is 10pM, 50pM, 100pM, 500pM The difference between the reactivity of SFB and the reactivity of SFB-modified sGV △ A was 0.081, 0.306, 0.644, 1.614, respectively, indicating that △ A increases depending on Goat anti-hCRP antibody concentration, hCRP membrane binding It was found that sGV and Goat anti-hCRP antibody specifically bound (FIG. 7).
Next, when the Goat anti-hCRP antibody concentration is kept constant at 500 pM, and the dilution ratio of anti-IgG antibody against Goat anti-hCRP antibody is 1: 100000, 1: 50000, 1: 20000, 1: 10000, ΔA is These were 0.350, 0.730, 1.218, and 1.849, respectively, indicating that ΔA increased depending on the anti-IgG antibody concentration (FIG. 8).
From the above results, it can be seen that LELIA using hCRP membrane-bound sGV can specifically detect anti-hCRP antibodies up to a concentration of about 10 pM, and the usefulness of LELIA using antigen membrane-bound sGV in the detection of specific antibodies is clarified became.

(2) Rabbit anti-hCRP antibody
We investigated the usefulness of LELIA in the specific reaction between Rabbit anti-hCRP antibody membrane-bound sGV and hCRP.
<Method> First, the sample tube was washed with PBS (pH 7.2), 1 ml of 3% Block Ace was added, and the mixture was incubated at 37 ° C. for 1 hour for blocking (blocking treatment). After blocking, the blocking solution was removed and washed with PBS. The prepared Rabbit anti-hCRP antibody membrane-bound sGV (Rabbit anti-hCRP antibody amount-sGV amount = 0.78 μg-30 nmol / 100 μl) sample, SFB-modified sGV (sGV amount 30 nmol) / 100 μl, control) or 100 μl each of 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer solution was added and centrifuged (19,500 × g, 20 min, 4 ° C.) (container adsorption step). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample tube (liposome washing step). Next, the supernatant was removed, 50 μl of hCRP solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (test substance binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample tube (test substance washing step). Further, the supernatant was removed, 50 μl of Goat anti-hCRP antibody solution diluted with PBS to a concentration of 5 nM was added and suspended, and incubated at 37 ° C. for 1 hour (primary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) 4 ° C.) to wash the sample tube (primary antibody washing step). Finally, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (secondary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample tube (secondary antibody washing step). After performing the same washing operation four times, suspend the precipitate in 0.5 ml of PBS, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour (transfer step), and centrifuge ( 19,500 × g, 20 min, 4 ° C.). After centrifugation, remove the supernatant, add 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ], and incubate at 37 ° C for 10 minutes for color development The reaction was stopped by adding 50 μl of 2N H ₂ SO ₄ , centrifuged (19,500 × g, 20 min, 4 ° C.), and the absorbance (A ₄₉₂ ) of the supernatant at 492 nm was measured using a microplate reader (Detection process).

<Results> First, the concentration of Goat anti-hCRP antibody is 5 nM, the dilution ratio of anti-IgG antibody as a secondary antibody against Goat anti-hCRP antibody is constant at 1: 10000, and the concentration of hCRP is 1 pM, 10 pM, 100 pM, The difference in reactivity between Rabbit anti-hCRP antibody membrane-bound sGV and SFB-modified sGV when 1 nM is 0.022, 0.091, 1.042, and 1.754, respectively, depending on the hCRP concentration. Rabbit anti-hCRP antibody membrane-bound sGV and hCRP were found to specifically bind (FIG. 9).
Next, the hCRP concentration was fixed at 1 nM, the Goat anti-hCRP antibody concentration was kept constant at 5 nM, and the dilution ratio of the anti-IgG antibody against the Goat anti-hCRP antibody was 1: 100000, 1: 50000, 1: 20000, 1: 10000 ΔA at that time was 0.187, 0.428, 0.932, and 1.603, respectively, indicating that ΔA increased depending on the anti-IgG antibody concentration (FIG. 10).
In addition, the concentration of Goat anti-hCRP antibody was 5 nM, the dilution ratio of anti-IgG antibody as a secondary antibody against Goat anti-hCRP antibody was fixed at 1: 5000, and the concentration of hCRP was 0.1 pM, 1 pM, 10 pM, 30 pM. ΔA at the time was 0.039, 0.048, 0.289, and 0.928, respectively, and hCRP could be detected in the concentration range of 1 pM to 10 pM (FIG. 11). Currently, there is a research report of detection sensitivity up to 20 pM by ELISA in the detection of hCRP (Non-patent Document 4), and an ELISA kit that can detect in the range of 1 pM-80 pM is sold (Non-patent Document 5). . In addition, the detection limit of hCRP by an electrochemical luminoimmunological test is 2 pM (Non-patent Document 6). This LELIA could detect hCRP with almost the same or higher sensitivity than these.
The above results indicate that LELIA using Rabbit anti-hCRP antibody membrane-bound sGV can specifically detect hCRP as a ligand, and the usefulness of LELIA using antibody membrane-bound sGV in ligand detection has been clarified. It was.

(3) Human insulin The usefulness of LELIA in the specific reaction between human insulin membrane-bound sGV and Rabbit anti-human insulin was examined.
<Method> First, after washing the sample tube with PBS (pH 7.2), 0.5 ml of 3% Block Ace (DS Pharma Biomedical Co., Ltd.) was added and incubated at 37 ° C. for 1 hour for blocking (blocking). processing). After blocking, remove the blocking solution and wash with PBS. Prepared human insulin membrane-bound sGV (human insulin amount-sGV amount = 1 μg-30 nmol / 100 μl) sample, SFB-modified sGV (sGV amount 30 nmol / 100 μl, control) or 10 mM 100 μl each of HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer was added and centrifuged (19,500 × g, 20 min, 4 ° C.) (container adsorption step). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample (liposome washing step). Next, the supernatant was removed, 50 μl of a Rabbit anti-human insulin antibody solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (primary antibody binding step). After the reaction, 0.5 ml of PBS was added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS was added to suspend the precipitate and centrifuged again (19,500 × g, 20 min, 4 ° C) to wash the sample tube (primary antibody washing step). Further, the supernatant was removed, 50 μl of an Anti-Rabbit IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (secondary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample (secondary antibody washing step). After performing the same washing operation four times, suspend the precipitate in 0.5 ml of PBS, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour (transfer step), and centrifuge ( 19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color is developed and 50 μl of 2N H ₂ SO ₄ is added to stop the reaction. Then, centrifugation (19,500 × g, 20 min, 4 ° C.) is performed, and the above is performed using a microplate reader (MULTISKAN JX, Thermo Fisher Scientific, Inc.). The absorbance (A ₄₉₂ ) of Kiyoshi at 492 nm was measured (detection step).

<Results> First, human insulin membrane binding when the dilution ratio of anti-IgG antibody to Rabbit anti-human insulin antibody is constant at 1: 2000, and the concentration of Rabbit anti-human insulin antibody is 0.2 nM, 0.5 nM, 1 nM, 2 nM The difference ΔA between the reactivity in the case of sGV and the reactivity in the case of SFB-modified sGV was 0.139, 0.251, 0.504, 0.749, respectively, indicating that ΔA increases depending on the Rabbit anti-human insulin antibody concentration, It was found that human insulin membrane-bound sGV and Rabbit anti-human insulin antibody were specifically bound (FIG. 12).
Next, the Rabbit anti-human insulin antibody concentration is kept constant at 1 nM, and the dilution ratio of the anti-IgG antibody against the Rabbit anti-human insulin antibody is 1: 20000, 1: 10000, 1: 5000, 1: 2000, 1: 1000, 1: ΔA at 500 was 0.021, 0.062, 0.175, 0.524, 1.004, and 1.404, respectively, indicating that ΔA increased depending on the anti-IgG antibody concentration (FIG. 13).
The above results show that LELIA using human insulin membrane-bound sGV can specifically detect anti-human insulin antibodies up to a concentration of about 0.2 nM, and the usefulness of LELIA using antigen membrane-bound sGV for specific antibody detection Became clear.

(4) Mouse anti-human insulin monoclonal antibody
We investigated the usefulness of LELIA in the specific reaction of mouse anti-human insulin monoclonal antibody membrane-bound sGV with human insulin.
<Method> First, the sample tube was washed with PBS (pH 7.2), 1 ml of 3% Block Ace was added, and the mixture was incubated at 37 ° C. for 1 hour for blocking (blocking treatment). Mouse blocking anti-human insulin monoclonal antibody membrane-bound sGV (Mouse anti-human insulin monoclonal antibody amount-sGV amount = 0.78 μg-30 nmol / 100 μl) sample, SFB-modified sGV (sGV) A volume of 30 nmol / 100 μl, control) or 100 μl of 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer was added and centrifuged (19,500 × g, 20 min, 4 ° C.) (container adsorption step). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample tube (liposome washing step). Next, the supernatant was removed, 50 μl of a human insulin solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (test substance binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample tube (test substance washing step). Further, the supernatant was removed, 50 μl of a Rabbit anti-human insulin antibody solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (primary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) 4 ° C.) to wash the sample tube (primary antibody washing step). Finally, the supernatant was removed, 50 μl of an Anti-Rabbit IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (secondary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample tube (secondary antibody washing step). After performing the same washing operation four times, suspend the precipitate in 0.5 ml of PBS, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour (transfer step), and centrifuge ( 19,500 × g, 20 min, 4 ° C.). After centrifugation, remove the supernatant, add 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ], and incubate at 37 ° C for 10 minutes for color development The reaction was stopped by adding 50 μl of 2N H ₂ SO ₄ , centrifuged (19,500 × g, 20 min, 4 ° C.), and the absorbance (A ₄₉₂ ) of the supernatant at 492 nm was measured using a microplate reader (Detection process).

<Results> First, the dilution ratio of Rabbit anti-human insulin antibody was fixed at 1: 500, the dilution ratio of anti-IgG antibody as a secondary antibody against Rabbit anti-human insulin antibody was constant at 1: 1000, and the insulin concentration was 0.1 pg / The difference ΔA between the reactivity of Mouse anti-human insulin monoclonal antibody membrane-bound sGV and the reactivity of SFB-modified sGV when ml, 1 pg / ml, 10 pg / ml, 30 pg / ml, 100 pg / ml is It was 0.109, 0.259, 0.444, 0.724, 0.990, respectively, indicating that ΔA increases depending on the human insulin concentration, and it was found that Mouse anti-human insulin monoclonal antibody membrane-bound sGV and human insulin specifically bind. (FIG. 14).
Next, the human insulin concentration is 10 pg / ml, the dilution ratio of the anti-IgG antibody, which is the secondary antibody against the Rabbit anti-human insulin antibody, is kept constant at 1: 1000, and the dilution ratio of the Rabbit anti-human insulin antibody is 1: 2000, 1 ΔA when: 1000, 1: 500, 1: 200 were 0.050, 0.229, 0408, 0.661, respectively, indicating that ΔA increases depending on the concentration of Rabbit anti-human insulin antibody (FIG. 15). .
In addition, the human insulin concentration is 10 pg / ml, the dilution ratio of Rabbit anti-human insulin antibody is constant at 1: 500, and the dilution ratio of anti-IgG antibody to Rabbit anti-human insulin antibody is 1: 5000, 1: 2000, 1: 1000 ΔA at 1: 500 was 0.058, 0.160, 0.374, and 0.805, respectively, indicating that ΔA increased depending on the anti-IgG antibody concentration (FIG. 16).
Currently, in the detection of human insulin, there is a research report of detection sensitivity up to 0.42 pmol / L by ELISA (Non-patent Document 4). Since the molecular weight of human insulin is 5,807, 0.42 pmol / L corresponds to about 2.4 pg / ml, and this LELIA was able to detect human insulin with higher sensitivity than this value.
The above results indicate that LELIA using Mouse anti-human insulin monoclonal antibody membrane-bound sGV can specifically detect human insulin as a ligand, and the usefulness of LELIA using antibody membrane-bound sGV in ligand detection is clear Became.

(5) Biotin
The usefulness of LELIA in the specific reaction between Biotin membrane-bound sGV and Goat anti-Biotin was investigated.
<Method> First, the sample tube was washed with PBS (pH 7.2), 0.5 ml of 3% Block Ace was added, and the mixture was incubated at 37 ° C. for 1 hour for blocking (blocking treatment). After blocking, the blocking solution was removed and washed with PBS. The prepared Biotin membrane-bound sGV (sGV amount 40 nmol / 100 μl) sample, SFB-modified sGV (sGV amount 40 nmol / 100 μl, control) or 10 mM HEPES-NaOH / 100 mM NaCl (pH 7) .5) 100 μl of each buffer solution was added and centrifuged (19,500 × g, 20 min, 4 ° C.) (container adsorption step). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample (liposome washing step). Next, the supernatant was removed, 50 μl of Goat anti-Biotin antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (primary antibody binding step). After the reaction, 0.5 ml of PBS was added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS was added to suspend the precipitate and centrifuged again (19,500 × g, 20 min, 4 ° C) to wash the sample tube (primary antibody washing step). Further, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (secondary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample (secondary antibody washing step). After performing the same washing operation four times, suspend the precipitate in 0.5 ml of PBS, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour (transfer step), and centrifuge ( 19,500 × g, 20 min, 4 ° C.). After centrifugation, the supernatant is removed, and 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ] is added and incubated at 37 ° C. for 10 minutes. Color development, stop the reaction by adding 50 μl of 2N H ₂ SO ₄ , centrifuge (19,500 × g, 20 min, 4 ° C.), and measure the absorbance (A ₄₉₂ ) of the supernatant at 492 nm using a microplate reader (Detection step).

<Results> First, in the case of Biotin membrane-bound sGV when the dilution ratio of anti-IgG antibody to Goat anti-Biotin antibody is fixed at 1: 5000 and the concentration of Goat anti-Biotin antibody is 1pM, 10pM, 100pM, 1nM The difference ΔA in reactivity with SFB-modified sGV was 0.004, 0.130, 1.672, and 3.189, respectively, indicating that ΔA increases depending on the Goat anti-Biotin antibody concentration, and biotin membrane binding It was found that sGV and Goat anti-Biotin antibody specifically bound (FIG. 17).
Next, when the Goat anti-Biotin antibody concentration is kept constant at 20 pM, and the dilution ratio of the anti-IgG antibody to Goat anti-Biotin antibody is 1: 10000, 1: 5000, 1: 2000, 1: 1000, ΔA is 0.119, 0.265, 0.499, and 0.666, respectively, indicating that ΔA increases depending on the anti-IgG antibody concentration (FIG. 18).
In addition, when the dilution ratio of the anti-IgG antibody, which is the secondary antibody against the Goat anti-Biotin antibody, is kept constant at 1: 1000, and the concentration of the Goat anti-Biotin antibody is 0.1 pM, 0.5 pM, 1 pM, 5 pM, 10 pM ΔA was 0.000, 0.007, 0.012, 0.146, and 0.305, respectively, and Goat anti-Biotin antibody was detected in the concentration range of 1 pM to 5 pM (FIG. 19). Tests using a photonic crystal microcavity sensor have reported that the detection limit of anti-Biotin antibodies is 20 pM (Non-patent Document 7), and this LELIA detects anti-Biotin antibodies with higher sensitivity. I was able to.
From the above results, it was found that the anti-Biotin antibody can be specifically detected by LELIA using Biotin membrane-bound sGV, and the usefulness of LELIA using antigen membrane-bound sGV in specific antibody detection has been clarified.

(6) Rabbit anti-Biotin antibody
We investigated the usefulness of LELIA in the specific reaction between Rabbit anti-Biotin antibody membrane-bound sGV and Biotin.
<Method> First, the sample tube was washed with PBS (pH 7.2), 1 ml of 3% Block Ace was added, and the mixture was incubated at 37 ° C. for 1 hour for blocking (blocking treatment). After blocking, the blocking solution was removed and washed with PBS. The prepared Rabbit anti-Biotin antibody membrane-bound sGV (Rabbit anti-Biotin antibody amount-sGV amount = 1 μg-30 nmol / 100 μl) sample, SFB-modified sGV (sGV amount 30 nmol / 100 μl, control) or 100 μl each of 10 mM HEPES-NaOH / 100 mM NaCl (pH 7.5) buffer solution was added and centrifuged (19,500 × g, 20 min, 4 ° C.) (container adsorption step). After centrifugation, the supernatant was removed, the precipitate was suspended in 0.5 ml of PBS, and centrifuged again (19,500 × g, 20 min, 4 ° C.) to wash the sample tube (liposome washing step). Next, the supernatant was removed, 50 μl of Biotin solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (test substance binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample tube (test substance washing step). Further, the supernatant was removed, 50 μl of Goat anti-Biotin antibody solution diluted to an arbitrary concentration with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (primary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) 4 ° C.) to wash the sample tube (primary antibody washing step). Finally, the supernatant was removed, 50 μl of an Anti-Goat IgG-HRP antibody solution arbitrarily diluted with PBS was added and suspended, and incubated at 37 ° C. for 1 hour (secondary antibody binding step). After the reaction, 0.5 ml of PBS is added and centrifuged (19,500 × g, 20 min, 4 ° C.). After removing the supernatant, 0.5 ml of PBS is added to suspend the precipitate and centrifuged again (19,500 × g, 20 min) , 4 ° C.) to wash the sample tube (secondary antibody washing step). After performing the same washing operation four times, suspend the precipitate in 0.5 ml of PBS, transfer the sample to another new tube that had been blocked with 3% Block Ace at 37 ° C for 1 hour (transfer step), and centrifuge ( 19,500 × g, 20 min, 4 ° C.). After centrifugation, remove the supernatant, add 100 μl of the substrate solution [0.1 M sodium citrate buffer (pH 5.2) + o-phenylenediamine (1 mg / ml) + 0.02% H ₂ O ₂ ], and incubate at 37 ° C for 10 minutes for color development The reaction was stopped by adding 50 μl of 2N H ₂ SO ₄ , centrifuged (19,500 × g, 20 min, 4 ° C.), and the absorbance (A ₄₉₂ ) of the supernatant at 492 nm was measured using a microplate reader (Detection process).

<Results> First, the concentration of Goat anti-Biotin antibody is 10 nM, the dilution ratio of anti-IgG antibody, which is a secondary antibody against Goat anti-Biotin antibody, is fixed at 1: 5000, and the concentration of Biotin is 0 fM, 0.5 fM, 1 fM. The difference ΔA between the reactivity in the case of Rabbit anti-Biotin antibody membrane-bound sGV and the reactivity in the case of SFB-modified sGV when 5 fM, 10 fM, 50 fM, 100 fM, 500 fM, 1 pM, 10 pM, 100 pM, 1 nM 0.000, 0.109, 0.167, 0.218, 0.345, 0.571, 0.659, 0.717, 0.762, 0.935, 1.093, 1.115, respectively, showing that ΔA increases depending on the concentration of Biotin, Rabbit anti-Biotin antibody membrane-bound sGV and Biotin was found to bind specifically (FIG. 20).
Next, ΔA when the biotin concentration is 10 fM, the dilution ratio of the anti-IgG antibody against the Goat anti-Biotin antibody is fixed at 1: 5000, and the Goat anti-Biotin antibody concentration is 0 nM, 0.4 nM, 2 nM, 10 nM. Were -0.001, 0.028, 0.079, and 0.345, respectively, indicating that ΔA increased depending on the Goat anti-Biotin antibody concentration (FIG. 21).
Furthermore, when the Biotin concentration is 10 fM, the Goat anti-Biotin antibody concentration is fixed at 5 nM, and the dilution ratio of the anti-IgG antibody to the Goat anti-Biotin antibody is 1: 10000, 1: 5000, 1: 2000, 1: 1000 The ΔA was 0.073, 0.151, 0.395, and 0.522, respectively, indicating that ΔA increased depending on the anti-IgG antibody concentration (FIG. 22).
From these results, it was found that LELIA using Rabbit anti-Biotin antibody membrane-bound sGV can specifically detect the ligand Biotin at a concentration of 0.5 fM. The detection limit of Biotin in the current ELISA is 4 pM (Non-patent Document 8), but the sensitivity of this LELIA was approximately 8000 times. Further, it has been reported that immunotin using gold nanoparticles can detect Biotin up to a concentration of 0.1 fM (Non-Patent Document 9), and this LELIA has almost the same detection sensitivity as compared with that. From the above, the usefulness of LELIA using antibody membrane-bound sGV in ligand detection became clear.
As described above, according to the present embodiment, LELIA (Liposome-based Enzyme-Linked ImmunoAssay), which is a novel measurement technique using a liposome capable of suppressing the background value lower than the ELISA method and lowering the measurement limit. ). According to LELIA, it was possible to quantify, identify, detect, identify, and search for a test substance with higher sensitivity than conventional ELISA methods.

Claims (6)

(1) a covalent binding step in which a test substance is covalently bonded to the surface of a liposome containing a saccharide-containing lipid thin film to form a test substance-bound liposome; (2) the test substance-bound liposome is added to a first container; (3) Liposome washing step for removing non-adsorbable test substance-bound liposomes, (4) In the liquid phase, the test substance-bound liposomes react with the primary antibody that recognizes the test substance. A primary antibody binding step for preparing a test substance / primary antibody-binding liposome, (5) a primary antibody washing step for separating the test substance / primary antibody-binding liposome and the liquid phase and removing an unnecessary liquid phase portion, (6) In the liquid phase, an enzyme-labeled secondary antibody that binds to the primary antibody is added to the test substance / primary antibody-binding liposome and reacted with the primary antibody, and the test substance / primary antibody is reacted. (7) Separating the test substance / primary antibody / enzyme-labeled secondary antibody-binding liposome from the liquid phase, and removing an unnecessary liquid phase portion. A secondary antibody washing step, (8) a transfer step of adding the solution and suspending the test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome, and then transferring the solution to a second container; and (9) An enzyme immunoassay method comprising a detection step of detecting by adding a substrate that can be detected by an enzyme reaction of an enzyme-labeled secondary antibody and reacting with an enzyme-labeled secondary antibody. (1) a covalent binding step in which an antibody recognizing a test substance is covalently bound to the surface of a liposome containing a saccharide-containing lipid thin film to form an antibody-bound liposome; (2) the antibody-bound liposome is added into a first container; A container adsorbing step for adsorbing to the wall of the container, (3) a liposome washing step for removing antibody-binding liposomes that cannot be adsorbed, and (4) a sample containing the test substance coexisting in the liquid phase. A test substance binding step for binding the antibody and the test substance to form an antibody / test substance binding liposome, (5) separating the antibody / test substance binding liposome and the liquid phase, and removing an unnecessary liquid phase portion. A test substance washing step to be removed, (6) in a liquid phase, reacting a primary antibody that recognizes the test substance with an antibody / test substance-bound liposome bound to the test substance, A primary antibody binding step for preparing a test substance / primary antibody-binding liposome; (7) a primary antibody washing step for separating the antibody / test substance / primary antibody-binding liposome from the liquid phase and removing an unnecessary liquid phase portion; In the liquid phase, an enzyme-labeled secondary antibody that binds to the primary antibody is added to the antibody / test substance / primary antibody-binding liposome and reacted with the primary antibody, to thereby react the antibody / test substance / primary antibody / enzyme-labeled secondary antibody. Secondary antibody binding step to form antibody-bound liposomes, (9) secondary antibody washing by separating the antibody / test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome from the liquid phase and removing unnecessary liquid phase portions Step, (10) adding the solution, suspending the antibody / test substance / primary antibody / enzyme-labeled secondary antibody-bound liposome, and then transferring the solution to a second container; and (11) enzyme label secondary By reaction with an enzyme in the body, it is reacted with an enzyme-labeled secondary antibody was added to substrate allowing detection, enzyme immunoassay, characterized in that it comprises a detection step of detecting. The enzyme immunoassay method according to claim 1 or 2, wherein the inner wall surface of the container is previously subjected to a blocking treatment with a blocking agent that prevents nonspecific adsorption. The enzyme immunoassay method according to any one of claims 1 to 3, wherein the solution used in the binding step and the washing step does not contain a surfactant. The enzyme immunoassay method according to any one of claims 1 to 4, wherein the liposome is a monolayer liposome or a multilamellar liposome. The said test substance is all the substances which can be couple | bonded with the said liposome containing a low molecular substance, a high molecular substance, a virus particle, and a microbial cell, The Claim 1 characterized by the above-mentioned. Enzyme immunoassay.