KR100560174B1 - Lateral flow quantitative assay method and strip and package therefor - Google Patents

Abstract

The present invention relates to a lateral flow quantitative assay method capable of highly quantitating one or more analytes. The present invention also relates to a lateral flow quantitative assay strip capable of simultaneously quantitating one or more analytes with high sensitivity and a package in which the strip and the laser induced surface fluorescence detection device of the present invention are integrally formed. The present invention can quantitate analytes with a lowest detection limit concentration of up to pg / ml. Thus, the present invention has the advantage of allowing simultaneous quantification of multiple analytes using simple lateral flow quantitative assay strips.

Analytes, Quantification, Strips, Surface Fluorescence

Description

Lateral flow quantitative assay method and strips and packages therefor {LATERAL FLOW QUANTITATIVE ASSAY METHOD AND STRIP AND PACKAGE THEREFOR}             

1 is a perspective view of a conventional lateral flow quantitative assay strip.

2 is a perspective view of a lateral flow quantitative assay strip as one embodiment according to the present invention.

3 is a top view of the conventional lateral flow quantitative assay strip shown in FIG. 1.

4 is a plan view of a lateral flow quantitative assay strip as one embodiment according to the invention shown in FIG. 2.

5 is a side view of a lateral flow quantitative assay strip as one embodiment according to the present invention shown in FIGS. 2 and 4.

6 is a side view of a lateral flow quantitative assay strip as another embodiment according to the present invention.

7 is a side view of a lateral flow quantitative assay strip as another embodiment according to the present invention.

8 is a side view of a lateral flow quantitative assay strip as another embodiment according to the present invention.

9 is a side view of a lateral flow quantitative assay strip as another embodiment according to the present invention.

10 is a side view of a lateral flow quantitative assay strip as another embodiment according to the present invention.

11 is a side view of a lateral flow quantitative assay strip as another embodiment according to the present invention.

12 is a block diagram of an elliptical reflector laser induced surface fluorescence detection device according to the present invention.

13 is a block diagram of a spherical laser-induced surface fluorescence detection device according to the present invention.

14 is a graph showing the minimum detection limit concentration measured for the surface antigen of the analyte using an ellipsoidal laser-induced fluorescence detection device according to the present invention (A: 4 ng / ml; B 400 pg / ml; C: 40 pg / ml).

15 is a graph comparing the results of PSA (prostate specific antigen) surface fluorescence intensity measured using an ellipsoidal laser laser-induced fluorescence detection device (A) and a conventional fluorescence detection scanner (B) according to the present invention. to be.

FIG. 16 is a graph showing the results of measurement and quantification of a plurality of test line strips using an elliptical reflector laser-induced fluorescence detection device according to the present invention.

Figure 17 is a schematic diagram of using the biotin-avidin system to the test line on the strip according to the present invention and the conventional method.

18 is a graph comparing the results quantified using the biotin-avidin system to the test line on the strip according to the present invention and the results quantified using the conventional method as a control.

19 is a graph showing the results of quantitative analysis by measuring the fluorescence intensity of the total PSA using the laser-induced fluorescence device of the present invention.

20 is a graph showing the results of quantitative analysis by measuring the fluorescence intensity of free PSA using the laser-induced fluorescence device of the present invention.

21 is a block diagram of a control means for a sample which is a component of the laser-induced surface fluorescence detection device according to the present invention.

<Description of the symbols for the main parts of the drawings>

Lateral Flow Quantitative Assay Strips (1) Sample Pads (2)

Binder Release Pads (3) Chromatography Media (4)

Absorption Pad (5) Support (6)

Adhesive (7) Wicking Pad (8)

First Standard Line (9) Second Standard Line (10)

Test Line (11) Top Laminate (12)

Second Chromatography Medium (13) Inspection Window (14)

Laser-Induced Fluorescence Detection Devices (20, 30)

Laser (21, 31) Sample (24, 34, 44)

Photodetector (27, 37) Excitation filter (22, 32)

Elliptical reflector (23) Spherical mirror (33)

Spatial filters (25, 35) Parallel optics (26, 36)

Fluorescence Filters (28, 38) Computers (29, 39)

Sample control means (40) Sample support (41)

Transport Means (42) Guides (43)

Motor (45)

The development of new diagnostic methods and diagnostic tools by crystallizing or quantifying trace substances in biopsies such as blood or urine has progressed rapidly over the past 30 years, and is still rapidly developing. Since the first introduction of radioimmunoassay (RIA) using radioisotopes in the 1950s, enzyme immunoassay (ELISA) was developed and developed in the 70s and 80s. ELISA immunoassay is now one of the most used methods and has become an essential tool in the research of medicine and life sciences. Recently, modified ELISA assays have been developed, one of which involves analyzing a large number of samples at once by immobilizing multiple antibodies in 96-wells.

In typical immunodiagnostic methods, including RIA and ELISA, one type of analyte can usually be quantified using a complex, multi-step, expensive laboratory analyzer. Therefore, it is not easy to use in a small hospital, emergency room, home, etc. which are not equipped with such a facility or facility. Diagnostic products designed to compensate for this weak point is a simple diagnostic kit using immunochromatography method.

The immunochromatography method can be used as a diagnostic kit to apply whole blood, serum, and urine biopsies to the designed device to confirm the test results within 15 minutes. Representative types of immunochromatography assays include lateral flow assays. Looking at the kit structure of the lateral flow analysis type, a sample pad to which a sample is applied, a release pad coated with a detection antibody, and a membrane for development in which the sample is moved and separated and an antibody antigen reaction occurs ( Predominantly nitrocellulose) or strips, and absorption pads for continuous movement of the sample. Detection antibodies are immobilized, for example, on colloidal gold particles to label detection. Latex beads or carbon particles may be used instead of gold particles. Diagnostic kits for lateral flow analysis are usually designed to detect analytes in the form of sandwiches. The analyte in the liquid sample is applied to the sample pad and begins to move, first developing in the form of an antigen-antibody conjugate by first reacting with a detection antibody that is unfixedly coated on the release pad. As it moves, it reacts once more with the capture antibody immobilized on the developing membrane to form a sandwich-type complex. Because the capture antibody is immobilized on the developing membrane, if antigen-antibody reactions continue to occur, accumulation of the complex takes place on the immobilized side of the capture antibody. Since proteins are transparent to the naked eye, the formation and relative amount of complexes are determined by the amount of gold particles attached.

The lateral flow analysis method is widely used in various fields such as pregnancy diagnosis, cancer diagnosis, microbial detection, and can be diagnosed very easily, but since the judgment is made with the naked eye, it is difficult to confirm the exact amount. In particular, it is not easy to accurately diagnose the case where the judgment should be made near the cut-off value. For example, in the case of prostate cancer, the cutoff value is 4 ng / ml, but the actual value is very similar, such as 3.9 ng / ml.

Immunodiagnosis is advancing rapidly, and in the near future, simpler and faster samples can be identified and analyzed to diagnose disease states. To date, quantifiable RIA or ELISA methods require several steps, such as processing and washing enzymes to quantify analytes in a sample. Similarly, conventional handy diagnostic kits are difficult to quantify. Therefore, it is time for more general methods to be faster, simpler, and more sensitive to quantification. This method should allow the untrained public to be diagnosed or analyzed anywhere.

In addition, conventional lateral flow quantitative assay strips, including all products known in the literature or actually on the market, have low sensitivity and are generally used as a means for crystallization rather than quantifying analytes. Only one or two types can be tested. In recent years, screening for disease usually involves dozens of analytes. In fact, the rapid development of molecular biology and medicine has led to an increasing number of analytes for disease testing. However, there are many kinds of analytes to be examined, and at present, it is a large burden on time and cost because of the situation of testing individual analytes separately. Under these circumstances, the rapid and accurate simultaneous quantification of many types of analytes is advantageous both economically and in many other ways, and the development of these products is increasingly eagerly demanded by the average consumer, including many medical personnel.

The inventors have developed a new lateral flow quantitative assay method that can simultaneously measure multiple types of analytes down to the lowest detection limit concentration of pg / ml, and strips for them, and a package incorporating these strips and laser-induced surface fluorescence detection devices. It was.

In one aspect, the present invention involves dropping a liquid sample that is expected to contain an analyte at one end of a chromatography medium so that the liquid sample is moved through the chromatography medium, and the analyte in the liquid sample is It reacts with labeled detectors adsorbed in compartments spaced at regular intervals in the direction of development from the loaded compartments along the chromatography medium to form a combination of analyte-labeled detectors, the combination of analyte-labeled detectors Moves between the chromatography media and reacts between the labeled and unlabeled captors by reacting with a trap that is the same or different and unlabeled and immobilized in a test window set at an intermediate position on the chromatography media. Labeled analyte-analyte, formed by trapping analyte into a sandwich To form a capture complex characters, and measuring the amount of complex formed accordingly in the lateral flow quantitative assay method for determining an analyte in a sample,

(a) the labeled detector is labeled with a fluorescent material to react with the analyte in the liquid sample to form a conjugate of the fluorescently labeled detector-analyte; (b) the unlabeled capturer was dispensed in a line to the inspection window on the chromatography medium to react with the conjugate that has migrated along the chromatography medium to form a complex of fluorescently labeled detector-analyte-unlabeled capture; ; (c) on a chromatography medium to which the fluorophore-labeled detector is adsorbed, a standard detector labeled with the same fluorophore as the detector and different from the detector and the trap and reacting with a standard in a liquid sample Unlabeled standard captures adsorbed together and reacted with the fluorescently labeled standard detector forward or backward relative to the inspection window on the chromatographic medium are either fixed as a single line or single in both front and rear. Fixed in lines to form a standard complex of fluorophore labeled standard detector-standards-unlabeled standard capture as the liquid sample moves along the chromatography medium; (d) irradiating light incident on the surface fluorescence medium of the composite and standard composite from the laser and passing through the excitation filter to focus the light reflected therefrom to the first focus of the ellipsoidal or spherical mirror at an appropriate size, The fluorescence emitted from the first focus and the scattered light of the incident light are reflected on the ellipsoidal reflector and focused at the second focus of the ellipsoidal reflector. The focused light is converted into parallel light by the parallel optical system, and the parallel light is converted into The lateral flow quantitative assay method is characterized in that the amount of analyte is determined by filtering the scattered light and injecting only pure fluorescent components into the photodetector to compare the amount of fluorescence of the complex with the amount of standard fluorescence represented by the standard complex. to provide.

As a second aspect, the present invention provides a backing, a sample pad adhered on one end of the support and into which a liquid sample is introduced, and one end overlapped at the end of the sample pad in the direction of the other end of the support. Conjugate releasing pads, wherein the labeled detectors are adhered to the support and react with the analytes in the sample to form a complex, and are immobilized at the ends of the labeled release pad toward the other end of the support. A chromatography medium in which one end is bonded to the support and the sample is developed and the same or different trappers are fixed to the detector which captures the complex with a binder and a sandwich that moves apart from the release pad to form a complex, and Unreacted material that absorbs the moving sample by chromatography and is also labeled A lateral flow quantitative assay strip comprising an absorption pad that absorbs and removes

Detectors adsorbed on the conjugated release pad non-fixedly are labeled with a fluorophore and further labeled on the conjugated release pad with the same fluorophore as the labeled fluorescent substance of the detector and react with a standard in a liquid sample. The standard detector is fixedly adsorbed; The capturer is fixed in a line in the inspection window on the chromatography medium; Different and unlabeled standard catchers from the detector and the catcher are either fixed in a single line as a standard line in front or rear of the inspection window or in a single line as a standard line in both front and rear of the inspection window; Standard complex of fluorophore-labeled detector-analyte-capture formed in the test window and standard complex of fluorophore-labeled standard-detector-standard-capture formed in the reference line as the liquid sample moves along the chromatography medium The surface fluorescence medium of is irradiated with the light incident from the laser and passed through the excitation filter, and the light reflected therefrom is focused to the first focus of the ellipsoidal or spherical mirror at an appropriate size and the fluorescence and incident light emitted at the first focus of the ellipsoidal mirror The scattered light is reflected by the ellipsoidal reflector and focused at the second focus of the ellipsoidal reflector. The focused light is converted into parallel light by the parallel optical system. The parallel light is passed through the fluorescence filter to filter the scattered light. The amount of fluorescence in the complex to the standard amount of fluorescence represented by the standard complex W provides a strip for determining the amount of the analyte by measuring the relative comparison.

As a third aspect, the present invention is directed to (i) a backing, a sample pad adhered on one end of the support and into which a liquid sample is introduced, and one end of the sample pad in the direction of the other end of the support. Conjugate releasing pads, the ends of which are adhered to the support and which are labeled with a labeled detector that reacts with the analyte in the sample to form a complex, and the conjugate release pad labeled toward the other end of the support. A chromatographic medium in which one or more ends are overlapped and adhered to the support, and the sample is developed, and the same or different trappers as the detectors are fixed by forming a complex by reacting with a sandwich and a binder moving apart from the release pad. unreacted and absorbed the sample moving by chromatography medium) and chromatography Comprising an absorption pad (absorption pad) to absorb and remove and quality; Detectors adsorbed on the conjugated release pad non-fixedly are labeled with a fluorophore and further labeled on the conjugated release pad with the same fluorophore as the labeled fluorescent substance of the detector and react with a standard in a liquid sample. The standard detector is fixedly adsorbed; The capturer is fixed in a line in the inspection window on the chromatography medium; A strip which is different from the detector and the trap and unlabeled standard catcher is fixed in a single line as a standard line in front or rear of the inspection window or in a single line as a standard line in both front and rear of the inspection window; (ii) consisting of a laser, an excitation filter, an ellipsoidal or spherical mirror, a control means for a sample emitting surface fluorescence, a parallel optical system, a fluorescence filter and a photodetector, these components comprising a liquid sample along the chromatographic medium of the strip. The surface fluorescent medium of the complex of the fluorescently labeled detector-analyte-capture formed in the inspection window and the standard complex of fluorescently-labeled standard detector-standard-standard capture formed in the reference line is incident from the laser while moving. Irradiated with the light passing through the excitation filter and reflected from it, Focused at the first focal point of the mirror or spherical mirror, the focusing point is located at the first focal point of the ellipsoidal reflector, and the fluorescence emitted from the first focal point and scattered light of the incident light are reflected by the ellipsoidal reflector to be the second focal point of the elliptical reflector. A laser-induced surface fluorescence detection device, which is focused on a parallel optical system and is transformed into parallel light and filters scattered light through a fluorescent filter and injects only pure fluorescent components into the photodetector, An analyte quantification package is provided so that the amount of fluorescence and the amount of standard fluorescence of the standard complex can be measured and relatively compared by the laser-induced surface fluorescence detection device to determine the amount of analyte in the liquid sample.

As used herein, the term "sensitivity" refers to the minimum threshold amount at which a complex of capturer, detector, and analyte can be detected.

As used herein, the term “epifluorescence” refers to a complex of fluorophore labeled detector-analyte-captures, each immobilized at the reference window and baseline of a lateral flow assay strip using chromatography and / or Or fluorescence emitted from a standard complex of fluorophore labeled standard detector-standards-standard captures.

The term “analyte” herein refers to the compound or composition to be analyzed in a liquid sample. Samples that can be used in the present invention can be selected from any sample containing the analyte, for example, physiological fluids such as urine, serum, plasma, blood, saliva, spinal fluid, ocular fluid, amniotic fluid, milk and wine Such as food, chemical waste streams such as food wastewater, and the like. Analytes that can be tested in the present invention can be divided into complete antigens and hapten (incomplete antigens). Here, the complete antigen refers to an antigenic substance that has the ability (immunogenicity) to induce antibody production by itself, and mainly contains a high molecular weight peptide hormone. The hapten refers to a substance that can bind to an antibody but is in itself incapable of inducing antibody production and includes peptides of relatively small molecular weight (less than about 1,000 molecular weights). The hapten acquires antibody producing capacity when bound to a protein such as, for example, bovine serum albumin.

In the present invention, complete antigens include, but are not limited to, the following:

(1) Examples of Peptide Hormones

1) Growth hormone (GH), corticosteroids (ACTH), melanocyte stimulating hormone (MSH), prolactin, thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH) and oxytocin Such as pituitary hormones;

2) calcium metabolism regulating hormones such as calcitonin and parathyroid hormone;

3) insulin, proinsulin and pancreatic hormones;

4) digestive tract hormones such as gastrin and secretin;

5) hormones that act on blood vessels such as angiotensin and bradykinin;

6) placental hormones such as human chorionic gonadotropin (hCG) and human placental lactogen (hPL);

(2) Examples of other substances

1) enzymes such as prostate acidic phosphatase (PAP), prostate-specific antigen (PSA), alkaline phosphatase, transaminase, lactic acid dehydrogenase (LDH), transaminase, trypsin and pepsinogen;

2) cancer-specific substances such as α-peptoprotein (AFP) and cancerous antigens (CEA);

3) serum protein components such as immunoglobulin G (IgG), fibrin-fibrinogen degradation products (FDP, D-dimer), antithrombin III (ATIII) and transferrin;

4) substances such as rheumatoid factor, serotonin, urokinase, ferritin and substance P.

In the present invention, haptens include, but are not limited to, the following:

(1) steroidal hapten

1) estrogens such as estrone, estradiol, estriol, estetrol, equilin and equilenin;

2) natural or synthetic luteohormones such as progesterone, pregnenadiol, pregnenatriol, 19-noretysterone and chloromadinone acetate;

3) male hormones such as testosterone, dehydroepiandrosterone, dihydrotestosterone, androsterone and thiocholoneron;

4) corticosteroids such as cortisol, cortisone, deoxycroticosterone, aldosterone and tetrahydrocortisol;

5) Vitamin D, cholesterol, cholic acid, deoxycholic acid and chenocholic acid and other steroids (eg cardiotonic steroids, saponins and sapogenins).

(2) physiologically active amines

1) catecholamines such as epinephrine, norepinephrine, dopamine and ephedrine and their metabolites;

2) physiologically active alkaloids such as morphine, codeine, heroin, morphine chloride, cocaine, mescaline, papaverine, narcotin, yohimbine, reserpin, ergotamine and strychinine;

3) amino group-containing psychotropic agents such as LSD, amphetamine, methanepetamine and meprobamate.

(3) another example

1) low molecular weight peptides without antigenicity such as TRH and LH-RH;

2) thyroid hormones such as diiodotyronine, triiodotyronine and thyroxine;

3) prostaglandins such as prostaglandin E2, prostaglandin E3 and prostaglandin F1a;

4) vitamins such as vitamin A, vitamin B (such as vitamins B1, B2, B6 and B12), vitamin E and vitamin K;

5) antibiotics such as penicillin, actinomycin, chloromycetin and tetracycline;

6) Other in vivo components and drugs and their metabolites administered into the organism.

In the present invention, the analyte may be specified as a single binding site (monoepitopic) ligand or a multiple binding site (polyepitopic) ligand. Polyvalent ligand analytes are usually poly (amino acids), ie polypeptides and proteins, polysaccharides, nucleic acids and combinations thereof. Combinations here include bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the like. In most cases, the polyvalent ligand analytes assayed in the present invention usually have a molecular weight of at least about 5,000, more usually at least about 10,000. The molecular weight of poly (amino acids) of interest in the poly (amino acid) category is generally about 5,000 to 5,000,000, more usually about 20,000 to 1,000,000. Of interest among hormones are usually molecular weights of about 5,000 to 60,000.

As an analyte in the present invention, several proteins can be considered as protein classes with similar structural features, protein classes with specific biological functions, protein classes associated with particular microorganisms, particularly pathogenic microorganisms, and the like. For cells and viruses, histocompatibility antigens or surface antigens are often of interest.

Proteins associated with the structure can be classified into protamine, histones, albumin, globulins, scleroproteins, phosphoproteins, mucoproteins, chromoproteins, lipoproteins, nucleoproteins, glycoproteins and proteoglycans. And other unclassified proteins such as somatotropin, prolactin, insulin and pepsin. All of these may be quantified in a package (which may be abbreviated herein as the package of the present invention) in which the lateral flow assay strip and the laser induced surface fluorescence detection device according to the present invention are integrally formed.

Many proteins found in human plasma are clinically important and all of them can be quantified in a package consisting of a lateral flow assay strip and a laser induced surface fluorescence detection device according to the invention. Examples of such plasma proteins are albumin-free, albumin, α ₁ - lipoprotein, α _1-acid glycoprotein, α _{1-antitrypsin} and α _{1-glycoprotein,} trans cor tin, 4,6S- post-albumin, tryptophan-poor α ₁ Glycoproteins and α ₁ X-glycoproteins, thyroxine-binding globulins, inter-α-trypsin-inhibitors, Gc-globulins (Gc 1-1, Gc 2-1 and Gc 2-2), haptoglobulins (Hp 1 -1, Hp 2-1 and Hp 2-2), serul to plasmin, choline esterase α ₂ - lipoprotein, myoglobin, C- reactive protein α ₂ - macroglobulin, α ₂ -HS- glycoprotein, Zn- α ₂ -glycoprotein and α ₂ -neuramino-glycoprotein, erythropoietin β-glycoprotein, transferrin, hemopexin, fibrinogen, plasminogen β ₂ -glycoprotein I and β ₂ -glycoprotein II, immune Globulin G (IgG), A (IgA), M (IgM), D (IgD), E (IgE) and the like.

Another example of an analyte that can be quantified using the package of the invention is the complement factor and coagulation factor. Complement factors are C'1, C'1q, C'1r, C'1s, C'2, C'3 (β ₁ A and α ₂ D), C'4, C'5, C'6, C ' 7, C'8 and C'9. Important blood coagulation factors include fibronogen, prothrombin, thrombin, tissue thromboplastin, proacerin, proacerin-promoting globulin, anti-hemorrhagic globulin (AHG), and Christmas factor (plasma thromboplastin component). , Steat-pro factor (autoprothrombin III), plasma thromboplastin antecedent, but factor (Hagemann factor), fibrin-stabilizing factor.

Important protein hormones that can be quantified using the package of the present invention include, but are not limited to, parathyroid hormone (parathrombin), tyrocalcitonin, insulin, glucagon, relaxine, erythropoietin, melanotropin, soma Peptide and protein hormones such as totrophin (growth hormone), corticotropin, tyrotropin, follicle stimulating hormone, luteinizing hormone, luteoma oriented hormone, gonadotropin (villi gonadotropin); Tissue hormones such as secretin, gastrin, angiotensin I and II, bradykinin, human placental lactogen; Peptide hormones derived from the pituitary gland, such as oxytocin, vasopressin, release factors (CRF, LRF, TRF, somatotropin-RF, GRF, FSH-RF, PIF, MIF).

Antigenic polysaccharides derived from microorganisms as analytes that can be quantified using the package of the present invention include, but are not limited to, Streptococcus pyogenes polysaccharides, Dilococcus pneumoniae polysaccharides, Neisseria meningitidis polysaccharides, Neisseria gonorrheae polysaccharides, Corynebacterium diphtheriae polysaccharides, Actinobacillus malay (Actinobacillus mallei) Tulla extracts (Francisella tularensis) polysaccharides and polysaccharides, Pasteurella pestis polysaccharides, Pasteurella multocida capsular antigen, Brucella abortus crude extract, Haemophilus influenza influenzae) polysaccharide, crude extract of Haemophilus pertussis , Treponema reiteri polysaccharide, Veillonella lipopolysaccharide, Erysipelothrix polysaccharide, Listeria monocytogenes polysaccharide, Chromobacterium polysaccharide , Brine extracts and polysaccharide fractions of Mycobacterium tuberculosis 90% phenol-extracted strains, Klebsiella aerogense polysaccharides, Klebsiella cloacae polysaccharides, Salmonella Salmonella typhosa polysaccharides and polysaccharides, Salmonella typhimurium polysaccharides, Shigella dysenteriae polysaccharides, Shigella flexneri and Shigella sonnei Crude extracts and polysaccharides, Rickettsiae crude extracts, Candida albicans polysaccharides and entamoeba histolytica ( Entamoeba histolytica) hemosensitin found in crude extracts.

Microorganisms quantified using the package of the present invention may be complete cells or dissolved, crushed or fragmented, and examples of the microorganisms include Corynebacteria, Corynebacterium diptheriae, Pneumococci, and Diple. Rococous pneumoniae, Streptococci, Streptococcus pyogenes, Streptococcus salivarus, Staphylococci, Staphylococcus Aureus, Staphylococcus aureus, Staphylococcus albus, Neisseriae, Neisseria meningitidis, Neisseria gonorrheae, Enterobacteria Enterobacteriaciae, Escherichia coli, Aerobacter aerogenes, Klebsiella pneumoniae siella pneumoniae, Salmonella typhosa, Salmonella choleraesuis, Salmonella typhimurium, Shigella dysenteriae, Shigella schmidz ai, Shigella schmidz Shigella arabinotarda, Shigella flexneri, Shigella boydii, Shigella Sonnei, Proteus vulgaris, Proteus mirabilis, Proteus Moteani (Proteus morgani), Pseudomonas aeruginosa, Alcaligenes faecalis, Vibrio cholerae, Hemophilus influenzae, Haemophilus ducrae (H. ducreyi, H. hemophilus, H. aegypticus, H. parainfluenzae, Bodetella pertussis, Pasteurella pestis (Pasteurella pestis), Pasteurella tulareusis, Brucella melitensis, Brucella abortus, Brucella suis, Bacillus anthracis, Bacillus Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Clostridium tetani, Crostridium perfringens, Crostridium perfringens Clostridium novyi, Clostridium septicum, Clostridium histolyticum, Clostridium tertium, Crossdium vi Clostridium bifermentans, Crostridium sporogenes, Mycobacterium tuberculosis hominis, Mycobacterium bovis, Mycobacterium avium Mycobacterium avium, Mycobacterium leprae, Mycobacterium paratuberculosis, Actinomyces israelii, Actinomyces bovis Tynomyces naeslundii, Nocardia asteroides, Nocardia brasiliensis, Spirochetes, Treponema Palidium Spyrillium Minus Spirillum minus, Treponema pertenue Streptobacillus, Treponema carateum, Borelia liqueur Bortislia recurrentis, Leptospira icterohemorrhagiae, Leptospira canicola, Mycoplasmas, Mycoplasmas pneumoniae, Listeria monocytose monocytogenes, Erysipelothrix rhusiopathiae, Strepptobacillus moniliformis, Donvania granulomatis, Bartonella bacilliformis, Bartonella bacilliformis Rickettsia prowazekii, Rickettsia mooseri, Rickettsia rickettsii, Rickettsia conori, Rickettsia australis, Rickettsia australis, Rickettsia sibiricus (Rickettsia sibiricus) Rickettsia akari, Rickettsia tsutsugamushi, Rickettsia burnetii, Rickettsia quintana, Chlamydia, Cryptococcus neoformans, Brastomyces dermatidis, Histoplasma capsulatum, Kotisidios (Coccidioides immitis), Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigatus, Mucor corymbifer (Absidia corymbifer) Rhizopus oryzae, Rhizopus arrhizus, Rhizopus nigricans, Sporotrichum schenkii, Fonsecaea pedrosoi, Fonsecaea pacata (Fonsecaea compacta), Fonseca dermatidis, Cladosporium carrionii, Phyalophora verrucos a), Aspergillus nidulans, Madurella mycetomi, Madurella grisea, Allescheria boydii, Pialospora truemay jeansilmei, Microsporum gypseum, Trichophyton mentagrophytes, Keratinomyces ajelloi, Microsporum canis, Trichophyton rubrum Trichophyton rubrum, Microsporum adnouini, Adenoviruses, Herpes Viruses, Herpes simplex, Varicella, Herpes Zoster Cytomegalovirus, Pox Viruses, Variola, Vaccinia, Poxvirus bovis, Paravacin cinia, Molluscum contagiosum, Picaornaviruses, Poliovirus, Coxsackievirus, Ecoviruses, Rhinoviruses ), Myxoviruses, Influenza A, B and C, Parainfluenza 1-4, Mumps Virus, Newcastle Disease Virus, Measles Virus ), Rinderpest Virus, Canine Distemper Virus, Syncytial Virus, Rubella Virus, Arboviruses, Eastern Equine Encephalitis Virus , Western Equine Encephalitis Virus, Sindbis Virus, Chikugunya Virus, Semliki Forest Virus, La Virus (Mayora Virus), St. Louis encephalitis virus (St. Louis Encephalitis Virus, California Encephalitis Virus, Colorado Tick Fever Virus, Yellow Fever Virus, Dengue Virus, Reoviruses 1 to 3 , Hepatitis A Virus, Hepatitis B Virus, Tumor Viruses, Rauscher Leukemia Virus, Gross Virus, Maloney Leukemia Virus ( Maloney Leukemia Virus, Epstein Barr Virus, Canine Heart Parasite (microfilaria), Malaria, Schistosomiasis, Coccidosis and Trichinosis. .

Monovalent ligand analytes that can be quantified using the package of the invention generally have a molecular weight of about 100 to 2,000, and more usually 125 to 1,000. Important analytes include drugs, metabolites, pesticides and contaminants. Important drugs include alkaloids. Examples of alkaloids include morphine alkaloids (eg morphine, codeine, heroin, dextromethorphan, derivatives and metabolites thereof), cocaine alkaloids (eg cocaine and benzoyl exonin, derivatives and metabolites thereof), ergot alkaloids (E.g. diethylamide of riseric acid), steroid alkaloids, aminazolyl alkaloids, quinazoline alkaloids, isoquinoline alkaloids, quinoline alkaloids (eg quinine and quinidine), diterpene alkaloids, derivatives and metabolites thereof Can be mentioned.

Analytes that can be quantified in the package of the present invention include steroid-based drugs. Examples thereof include estrogens, androgens, andocortical steroids, bile acids, cardiac glycosides and aglycones (eg, digoxin and digoxigenin, saponins and sapogenins, derivatives and metabolites thereof). Also included are steroid mimics such as diethylstilbestrol. Still other drugs include lactams having 5 to 6 reductions, examples of which include barbiturates (eg phenobarbital and secobarbital), diphenylhydantoin, pyrimidone, etosuccimid and their metabolites Can be mentioned. Other drugs include aminoalkylbenzenes having 2 or 3 carbon atoms of alkyl, examples of which include amphetamines, catecholamines (e.g. ephedrine), L-dopa, epinetin, narcin, papaverine and their metabolism. Water is available. Still other drugs include benzheterocyclics in which the heterocyclic rings are azepins, diazepines and phenothiazines, examples of which are oxazepam, chlorpromazine, tegestol, imipramine, derivatives and metabolites thereof. Can be mentioned. Still other drugs include purines and examples include theophylline, caffeine, metabolites and derivatives thereof. Still other drugs include substances derived from marijuana, and examples thereof include cannabinol and tetrahydrocannabinol. Still other drugs include vitamins such as A, B (eg B ₁₂ ), C, D, E, K, folic acid and thiamine. Still other drugs include prostaglandins, which differ in their degree and site of hydroxylation and unsaturation. Still other drugs include antibiotics, examples of which include penicillin, chloromycetin, antinomycetin, tetracycline, theramycin, metabolites and derivatives thereof. Still other drugs include nucleosides and nucleotides, examples of which include ATP, NAD, FMN, adenosine, guanosine, thymidine and cytidine with appropriate sugar and phosphate substituents. Other drugs that may not be classified include: methadone, meprobamate, serotonin, meperidine, amitriptyline, nortriptyline, lidocaine, procaineamide, acetylprocaine Amides, propranolol, griseofulvin, valproic acid, butyrophenone, antihistamines, anticholinergic drugs (eg atropine), metabolites and derivatives thereof. Metabolites associated with disease states include spermine, galactose, phenylpyruvic acid and type 1 porphyrin. Still other drugs include aminoglycosides and examples thereof include gentamicin, kanamycin, tobramycin and amishcin.

Analytes that can be quantified in the present invention include insecticides, and examples thereof include polyhalogenated biphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenated sulfenamides, metabolites and derivatives thereof.

Analytes that can be quantified using the package of the invention include receptor analytes. In this case the molecular weight is generally from 10,000 to 2 x 10 ⁸ , more usually from 10,000 to 10 ⁶ . For immunoglobulin IgA, IgG, IgE and IgM, the molecular weight is generally from about 160,000 to about 10 ⁶ . Enzymes usually have a molecular weight of about 10,000 to 1,000,000. Natural receptors vary in molecular weight and generally are at least about 25,000 and can be at least 10 ⁶ , including, for example, avidin, DNA, RNA, thyroxine binding globulin, thyroxine binding prialbumin, transcrotin and the like.

In addition to the analytes described above, the package of the present invention can be used to quantify tumor markers, angiogenesis related markers, heart disease markers, Alzheimer's disease related markers, cancer related genes, environmental toxins, drugs of abuse, and the like. Tumor markers include, but are not limited to, alpha 1-acidglycoprotein, CEA, AFP, PSA / free PSA, CA 15-3, CA 19-9, CA 27-9, CA 50, CA 125, CA 72- 4, calcitonin, elastase-1, ferritin, pepsinogen I, PIVKA II, procollagen III peptide, beta HCG, beta 2-microblobulin, neuron specific enolase, CYFRA 21-1 (psychokeratin 19) , Secretin, NMP (nuclear matrix protein), COX-1 and TPA (tissue polypeptide antigen). Angiogenesis related markers include angiogenic factors and hemostatic factors. Examples of angiogenic factors include aFGF (acidic fibroblast growth factor), bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), angiogenin, angiopoietin 1, heparanase, scattering factor (scatter) factor), HGF (hepatocyte growth factor), PDGF (platelet derived growth factor), iotropin, TGF alpha, TGF beta, IL-8, TNF alpha and prostaglandin E1 and E2. Examples of hemostatic factors include endostatin, angiostatin, cartilage derivative inhibitors, heparanase, angiopoietin 2, IFN alpha, IFN beta, IFN decay, platelet factor 4, 16 kDa prolactin fragment, protamine, thrombospandine, TIMP (metalloph Tissue inhibitors of rotase), thalidomide, and TNP470 (fumazilin analogue). Examples of heart disease markers include creatine kinase-BB, creatine kinase-MB, creatine kinase-MM, myoglobin, MLC (myosin light chain), troponin I, troponin C, troponin ITC, troponin T, CRP, FABP (fatty acid Binding proteins). Examples of Alzheimer's disease associated markers include glutamine synthetase, melano transferrin, beta-amyloid protein. Examples of cancer-associated genetics include bcl-2, C-erbB-2, C-myc, CSF-1 receptor, EGF receptor, H-ras, K-ras (p12), L-myc, mdr-1, N- myc, N-ras, p53 exon 4, p53 exon 5, p53 exon 6, p53 exon 7, p53 exon 8, p53 exon 9, TcR-α, TcR-β, TcR-γ, TcR-δ. Examples of environmental toxins include microcystine, dioxin and PCB. Drugs of abuse include amphetamine, barbiturate, benzodiazepines, cannabinoids, cocaine, morphine, phencycline, TBPE and the like.

In the present invention, a fluorescent substance is specifically used as a label that can serve as an index for the presence or absence of an analyte in a liquid sample. As the fluorescent material, the absorption wavelength and the emission wavelength may be different from each other by more than 20 nm. Representative fluorescent materials include, but are not limited to, fluorescence particles, quantum dots, lanthanide chelates (eg, samarium (Sm), europium (Eu), and terbium (Tb)) And fluorescence (eg, FITC, rhodamine green, thiadicarbocyanine, Cy2, Cy3, Cy5, Cy5.5, Alexa 488, Alexa 546, Alexa 594 and Alexa 647). Preferred fluorescent materials used to detect DNA are Cy3 and Cy5. In general, the intensity of fluorescence is directly proportional to the intensity of excitation light.

In the present invention, the label is bound to a detector that specifically binds to the analyte through a linker. Such linkers include, but are not limited to, N- [k-maleimidoundocanoyloxy])-sulfosucciamide ester (sulfo-KMUS), succinimidyl-4- [N-maleimidomethyl] -cyclo Hexane-1-carboxy [6-amidocaprorate] (LC-SMCC), NK-maleimidododecanoic acid (KMUA), succinimidyl-4- [p-maleimidophenyl] butyrate (SMBP), succinimi Diyl-6-[(β-maleimido-propionamido) hexanoate] (SMPH), succinimidyl-4- [N-maleimidomethyl] -cyclohexane-1-carboxylate (SMCC) Posuccinimidyl-4- [N-maleimidomethyl] -cyclohexane-1-carboxylate (sulfo-SMCC), N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB), sulfostone Cinimidyl [4-iodoacetyl] aminobenzoate (sulfo-SIAB), N- [γ-maleimidobutyryloxy] sulfo-succinimide ester (sulfo-GMBS), N- [γ-maleimidobuti Ryloxy] succinimide ester (GMBS), succinimidyl -3- [bromoacetamido] propionate (SBAP), N-β-maleimidopropionic acid (BMPA), N- [α-maleimidoacetoxy] succinimide ester (AMAS), N-succinimide Dill S-acetylthiopropionate (SATP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS) Ne-maleimidocaprylic acid (EMCA), N- [e-maleimidocaproyloxy] succinimide ester (EMCS), N-succinimidyl- [4-vinylsulfonyl] benzoate (SVSB), N- [β-maleimidopropyloxy] succinimide ester (BMPS) and 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC). The linkers will react with the thiol group of the detector, for example.

The lateral flow assay strips according to the present invention may take the form of rectangles, circles, ovals, triangles, and many other shapes, provided that there is at least one direction in which the test solution can be moved by capillary forces. There may be other directions of movement, such as oval or circular, in which the test solution is in contact in the middle. However, it should be considered that the test solution must be moved to a predetermined position in at least one direction. The thickness of the strip according to the invention is not critical but is usually 0.1 to 2 mm, more usually 0.15 to 1 mm, preferably 0.2 to 0.7 mm. In general, the minimum thickness is determined by the strength of the strip material and the need to generate an easy detection signal while the maximum thickness is determined by the ease of handling of the reagent and the cost of the reagent. In order to preserve the reagents and provide a sample of defined size, the width of the strip is generally made relatively narrow, usually less than 20 mm, preferably less than 10 mm. In general, the width of the strip should not be less than about 1.0 mm and the usual range is about 2 mm to 12 mm and the preferred range is about 4 mm to 8 mm. The length of the strip is determined by considering the type of analyte, the number of test or point and standard lines on the chromatography medium, the spacing between the pads, and the ease of handling. It is usually 1 to 40 cm, preferably about 2 to 25 cm, more preferably about 4 to 20 cm. However, the strip can be of virtually any length.

The solvent for the liquid sample to be analyzed is usually an aqueous medium, which is an oxidation solvent having 1 to 6 carbon atoms, more usually 1 to 4 carbon atoms, including up to about 40% by weight of other polar solvents, in particular alcohols, ethers, etc. It may include. Usually, cosolvent is present at less than about 20 weight percent. Depending on the nature of the sample, some or all of the aqueous medium may be provided by the sample itself under some circumstances.

The pH for the medium is usually 4 to 11, more usually 5 to 10, preferably 6 to 9. The pH is chosen to maintain an important binding affinity site of the binding elements and any generation of the signal by the signal generating system. Various buffers can be used to adjust and maintain the desired pH during the assay. Representative buffers include borate, phosphate, carbonate, tris, barbital. Although the particular buffer used is not critical, one buffer may be preferred over the other in individual assays. Preferably about 0.05 to 0.5% by weight of nonionic detergent is included in the sample. Various polyoxyalkylene compounds of about 200 to 20,000 daltons can be used.

Mild temperatures are usually used to perform the assay, and preferably substantially constant temperatures are used. The temperature for generating the assay signal is generally about 4 ° C. to 50 ° C., more usually about 10 ° C. to 40 ° C., and often at ambient temperature, ie, about 15 ° C. to 25 ° C.

The concentration of the analyte to be assayed in the aqueous test solution is generally about 10 ^-4 to about 10 ^-15 M, more usually about 10 ^-6 to 10 ^-14 M. The concentration of other reagents is usually determined taking into account such as the concentration and protocol of the desired analyte.

The concentrations of many different reagents in the sample and reagent solutions are generally determined by the concentration range of the desired analyte, while the final concentration of each reagent is determined by experience to optimize the sensitivity of the assay in the desired range. Individual reagents with specific protocols can be used in excess unless the sensitivity of the assay is degraded.

Hereinafter, a package in which the lateral flow assay strip and the laser-induced surface fluorescence detection device according to the present invention are integrally formed will be described.

Backing of lateral flow assay strip

The supports are usually water insoluble, nonporous and rigid and usually have the same length and width of the pads that develop the sample above them, but may be larger or smaller. A wide variety of natural and synthetic organic and inorganic materials may be used provided the support should not interfere with the capillary action of the absorbent material, bind nonspecifically with the analyte, or interfere with the reaction of the analyte and the detector. Representative polymers include, but are not limited to, polyethylene, polyester, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), nylon, poly (vinyl butyrate), glass, Ceramics, metals and the like.

The support is usually coated with adhesive to which various pads are attached. Choosing the right adhesive can help improve strip performance and extend its life. Pressure-sensitive adhesives (PSA) are representative for use in lateral flow assay strips according to the present invention. Bonding of various pads in a typical lateral flow assay strip is accomplished by adhesive penetration into the pores of their pads and thus the pads bonding with the support. This process of moving the adhesive under normal conditions is called cold flow. Since the PSA is not heated in the lamination process, some cold flow is necessary for the bond between the pad and the support to be formed. If the degree of cold flow is too low, the initial bonding force may be low, resulting in improper mating of the pad and the support. Conversely, if the degree of cold flow is too high, the adhesive may move into the pads that are held together, especially during the storage period of the strip, which may cause pore blocking, hydrophobic staining, or rewetting of the pads. The problem associated with cold flow of such adhesives can be solved by using direct-casting membranes. For example, these membranes prevent the adhesive from entering the pores of the membrane by the supporting plastic sheet, thus preventing the adhesive from moving up and down during storage.

Sample pad of lateral flow assay strip

The sample pad basically serves to receive a sample containing the analyte. In addition to this function, the sample pad may have a function of filtering insoluble particles in the sample. In view of this, the sample pad of the present invention is preferably a cellulose filter paper or a glass fiber filter paper which is a material to which the filtration function is added. Generally, S & S's cellulose membranes (grade 903) are used.

The sample pad prevents nonspecific adsorption of the analyte in the sample to its material, as well as assists the components of the sample to move easily through the chromatographic medium, maintains the sensitivity of the reaction, and provides label-labeled detectors and samples. Pretreatment is preferred to prevent as much as possible of the unwanted nonspecific reactions between components. Pretreatment of the sample pad is usually carried out by treating the sample pad with an inert protein or by treating with a surfactant. Pretreatment with inert proteins may include, for example, pads containing 0.1-10% bovine serum albumin (BSA) -containing 0.1 M Tris buffer (pH 6-9), 0.1% to 10% in 0.1 M Tris buffer (pH 6-9). It is performed by immersing in a solution of skim milk and / or 0.1% to 10% casein solution and leaving it at 37 ° C. for 1 hour or 4 hours at 24 ° C. and then washing the pad with Tris buffer and then drying. Pretreatment with a surfactant is carried out, for example, by immersing the pad in a solution containing 0.01% to 1% of the nonionic surfactant Triton X-100 or Tween 20 and then drying. Preferred pretreatment is treatment with an inert protein and also with a surfactant. However, the determination of this pretreatment will depend on the type of analyte and sample.

Conjugate releasing pad of lateral flow assay strip

The conjugate release pad is immobilized without a phosphor-labeled detector capable of reacting with the analyte in the sample to form a conjugate. The detector is immobilized and adsorbed, thus forming a conjugate by reaction with the analyte in the sample and then moving together as the sample evolves through the chromatography medium.

The material for the binder release pad is preferably to provide good particle retention with fast filtration rates. As such, synthetic materials such as polyester and glass fiber filters can be used. Generally, glass fibers and polyesters from S & S are used. Because they are biologically inert and have more sophisticated fibers than natural materials, they do not twist or swell when aqueous reagents or samples are added. Preferably, the conjugate release pad is pretreated with a reagent such as a surfactant to facilitate release and migration of the conjugate while preventing the analyte and fluorophore-labeled detector from adsorbing nonspecifically.

The method of adsorbing the reagent to the conjugate release pad includes an impregnation process of dipping and drying a pad, such as glass fibers, in a specifically formulated high density reagent solution. The immersion method is simple but has some problems. The first is that they can be wrinkled or twisted while the pads are drying. The second is that while the pad is drying in the oven, depending on the pad's position, the reagent may dislodge or reconstruct due to surface tension and gravity effects. Third, chemical changes can occur over time in the pad immersion bath, resulting in different adsorption rates between different reagents, resulting in uneven coating of the reagents on the pad. One way to minimize this problem is to dry the pads over several hours in an oven below 40 ° C. Another method is to lyophilize instead of drying in an oven. Such lyophilization is preferable to drying in an oven in that the stability of the detector can be secured.

As an alternative to the dipping method, a dispensing process is used. This method uses a dispenser to dispense 12-15 μl of reagent solution per cm of pad, followed by drying. Drying the pad is carried out in the same manner as the dipping method and can also be lyophilized.

As another aspect, stabilizer and blocker may be used in the binder release pad. Examples of stabilizers include sugars such as sucrose, trehalose. Blocking agents include, but are not limited to, proteins such as BSA (bovine serum albumin), gelatin, casein, skim milk and the like.

Chromatography medium of lateral flow assay strip

The chromatography medium preferably can be any and preferably has uniform properties, as long as the liquid sample and the binder can be rapidly moved by capillary forces to reach the trappers immobilized thereon. Generally, the chromatography medium refers to a porous material that is easy to move by the aqueous medium in response to capillary forces and has a pore size of at least 0.1 μm, preferably at least 1.0 μm. Such materials can generally be hydrophilic or hydrophilic and include, for example, inorganic powders (eg, silica, magnesium sulfate, and alumina); Natural polymeric materials, in particular cellulosic materials and materials derived from cellulose (eg, fiber-containing paper such as filter paper, chromatography paper, etc.); Synthetic or modified natural polymers (eg, nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, crosslinked dextran, agarose, polyacrylate, etc.). The above exemplified materials may be used alone or in combination with other materials. Another example is a ceramic material. The chromatography medium can be bound to the support. As another alternative, the chromatography medium may itself be a support. The chromatography medium may be multifunctional or modified to be multifunctional to enable covalent binding of the capture.

Chromatography media is preferably used when the high concentration of capture chemically bound to the chromatography medium is used to capture and react with a combination of analytes and detectors that have migrated from the binder pad. . When CNBr activated cellulose is selected and used as the material of the medium, the activated cellulose filter paper can be obtained from Ceska and Lundkvist, Immunochemistry, 9, 1021 (1972) and Lehtone, Viljanen et al., J. Immunol. Methods, 36, 63 (1980) and 34, 61 (1980) of the same document, can be readily prepared by known methods. Alternatively, when the material is selected and used DBM activated cellulose can be easily prepared by known methods such as those described in Alwine, Methods Enzymol., 68, 220 (1979). In addition, commercially available activated nylon films (Pall Immunodyne, USA) can be used.

Chromatography media are important for their ability to fix the trap. This binding capacity depends on the pore structure of the medium and the post-treatment of the medium. Chromatography media that can preferably be used in the present invention are nitrocellulose (NC) membranes, examples of which are described in Table 1 below.

Table 1

manufacturer product name Sec / 4cm (flow rate ^(a) ) IgG / cm ^{2 (b)} S & S (unsupported) AE 98 160-210 20 ~ 30ug AE 99 120-160 20 ~ 30ug AE 100 90-120 20 ~ 30ug Millipore (with support) HF 090 80-100 > 95 HF 120 107-133 > 95 HF 135 120-150 > 95 HF 180 160-200 > 95 HF 240 214-266 > 120 Sartorius (with combined support) CN 90 88-94 10-30 CN 140 137-153 10-30 CN 200 205-233 10-30

(a) The time it takes for distilled water to rise 4cm in the medium

(b) Binding capacity-amount of IgG attachable per square centimeter of medium.

Preferred chromatography media is the CN 90 membrane. The CN 90 membrane has the narrowest range of variation in flow rate variation of +/- 3 seconds among the above families. Binding capacity is enough to about 10 to 30ug because of the excellent amplification of the fluorescent label. Above all, the greatest advantage of this membrane is its excellent reproducibility of lateral flow velocity.

The trap is chemically bound to and fixed on the chromatography medium. Such chemical bonding may be carried out according to known methods (LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Volume 15, Edited by R. H. BURDON and P. H. Van KNIPPENBERG ELSEVIER AMSTERDAM: NEW YORK, OXFORD (1985) P. 318-322). The capture may also be bound via a second substance (eg, antibody protein) on the chromatography medium. If the second substance is an antibody (second antibody) and the trap to be immobilized is a monoclonal antibody derived from a mouse, an appropriate amount of capture (monocle) after the excess anti-mouse yG (gamma globulin) hetero-animal antibody is bound Activated paper sheets can be used, in which a ronal antibody) is bound by an immune response. If the second substance is a protein, for example, an activated paper sheet may be used in which an excess amount of protein A is bound followed by an appropriate amount of capture antibody.

Blocking technology is used as a method to uniformly wet the test line in the inspection window on the chromatographic medium. Blocking the chromatography medium with a material that enhances the rewetting of the chromatography medium ensures a rapid and uniform rewetting of the medium. There are four types of blocking agents used: proteins (eg BSA and gelatin), surfactants (SDS, Tween 20 and Triton X-100), polymers (PVA, PEG and PVP). Blockers can be used at three points. The first is the application of blockers to the chromatography medium. While this method can provide a very uniform rewetting effect, the medium must be blocked after application of the trap but prior to the attachment of the sample pad, requires expensive coating equipment and needs to redissolve the trap and the blocker itself Can reduce the antigenicity and shelf life of the capture. The second is to incorporate the blocking agent into the sample pad or the conjugate release pad. This method is cheap and easy to implement, and there is no need to re-dissolve the catcher. This method is preferred in terms of ease of handling, although its blocking efficiency is lower than that of the first method. Third is the inclusion of a blocker in the trapper application buffer. This method is also inexpensive and easy to implement, but has a poor blocking effect, and the inclusion of a blocking agent decreases the capture's antigenicity and / or shelf life and is likely to spread.

Two methods can be used to prevent nonspecific binding of reagents used in sample pads, binder release pads and chromatography media. One method is to block the nonspecific binding site on the chromatography medium by applying the trapper to the chromatography medium by dipping or spraying it with a solution containing a protein or highly polar polymer (eg polyvinyl alcohol). However, this technique has the potential to displace the trap, particularly if the trap is not optimally fixed by complete drying prior to the blocking step. Another method is to add a blocker to the sample pad. The blocking agent may not at least initially prevent the capturer from binding to the chromatography medium. When the liquid sample is loaded onto the sample pad, the blocking agent is redissolved on the sample pad and moves with the sample. Ideally, a sufficient amount of blocking agent is added to the sample pad to prevent nonspecific binding of both the analyte and the detector present in the sample.

The present invention utilizes a biotin-avidin conjugate as a further preferred embodiment. That is, the avidin is immobilized by immobilizing the capturer (e.g., antibody or antigenic protein) without dispensing the test line on the strip. Instead, biotin is attached to the trapper so that the biotin binds to avidin so that the trapper can be automatically detected at the test line on the strip. When binding the biotin to the capturer, the biotin is specifically bound to a specific site of the protein by using an amine group of lysine or arginine among amino acids when the capturer is a protein. Biotin-bound protein is distributed on the strip to specifically bind to immobilized avidin, and always maintains the same orientation. Adsorption of biotin is not due to adsorption. little. For this reason, unlike the conventional non-directionally immobilized on the membrane of the strip without biodirectionality, the biotin-avidin method is always immobilized in a fixed direction in the capture protein has the same directionality, there is no change in structure or function It can react more effectively with the analytes present in the sample. Thus, the biotin-avidin method can maintain higher sensitivity at the same analyte concentration. High sensitivity can be maintained even if the concentration is at least 10-100 times lower than the concentration of the immobilized material. In addition, the use of avidin-dispensed strips does not require the preparation of a new type of strip even if the analytes are different. That is, avidin strips can be commonly used. However, by preparing only different protein-biotin conjugates and protein-fluorescent conjugates as detectors, various measurement items can be tested. The method can be prepared using the conjugate pad in the same manner as the conventional method, and can also be reacted with the analyte in solution without preparing the conjugate pad to directly react the analyte and protein-biotin conjugate and protein-fluorescence on the sample pad without the conjugate pad. Experiments can also be performed by mixing the binders together. Fig. 17 is a schematic diagram of using the biotin-avidin system on the test line on the strip and using the conventional method.

Absorption pad of lateral flow assay strip

Absorption pads are means for physically absorbing samples that have migrated by capillary chromatography and removing unreacted materials. That is, the absorption pad is set at the end of the lateral flow assay strip and acts as a pump or reservoir to control and promote and contain the rate of sample and reagent that is traveling along the chromatography medium. The rate of movement of the samples and reagents may vary depending on the quality and size of the absorbent pad and commonly used absorbent pads are those formed from water-absorbing materials such as cellulose filter paper, nonwovens, cloth or cellulose acetate.

Wicking pad of the lateral flow black strip

It is used for the purpose of promoting the diffusion of a sample such as blood and at the same time preventing bacterial contamination of the binder pad during storage of the strip. Generally, AW14-20T4 from Pall Corporation, USA is used and its material is hydroxylated polyester.

Lateral flow assay strips in accordance with the present invention will be more specifically illustrated with reference to the accompanying drawings. From these examples the features and advantages of the invention will be apparent.

1 and 3 show a conventional lateral flow assay strip. From Fig. 1, the conventional lateral flow assay strip 1 has a sample pad 2 into which a liquid sample containing an analyte is introduced at one end of the support via an adhesive layer on the support 6, followed by a support 6 The binder release pad 3, the chromatography medium 4 and the absorbent pad 5 are attached in the opposite distal direction of the &lt; RTI ID = 0.0 &gt; On the binder release pad 3, a labeled detector capable of reacting with an analyte in the liquid sample to form a conjugate while the liquid sample is chromatographically moved by capillary action is adsorbed non-fixedly. On the chromatography medium (4), the same or different trappers as the detector are dispensed to be fixed on the medium (4) by chemical bonding to one line (test line) 11, which capture line 11 is The liquid sample and the binder formed in the binder release pad 3 chemically react as they move chromatographically to capture the binder to form a complex of labeled detector-analyte-captures. The remaining unreacted material and liquid sample are continuously chromatographically absorbed by the absorbent pad 5 attached to the end of the support. The amount of analyte is determined as the amount of the complex, and the amount of the complex determines the luminescence intensity of the complex immobilized on the chromatography medium, which is labeled the same as the detector in advance and differs from the detector and the capture by this standard detector. The analyte is quantified by quantifying the standard luminescence intensity and calculating it as a relative value for the complex formed by reacting different and unlabeled standard capture with the detector.

As mentioned above, conventional lateral flow assay strips are designed to quantify only one type of analyte in a biological sample.

2, 4 and 5 show lateral flow assay strips as one embodiment of the present invention. This lateral flow assay strip unfixedly adsorbs four or more detectors on the binder release pad 2 and captures the same number of traps as the detectors in the inspection window 14 on the chromatography medium 4. By dispensing the array by the fine test line 11 of the multiple analytes can be simultaneously tested. Each type of trap is divided into individual test lines so that the number of test lines is the same as the number of catches, so that multiple analytes can be tested simultaneously. The number of test lines may be limited by the size of the lateral flow assay strip, the type and amount of reagents (detectors and capturers) used in the strip, sensitivity, and the like. The number of test lines used in the lateral flow assay strips according to the invention is generally 2-20, preferably 5-15.

6 shows a lateral flow assay strip as another embodiment according to the present invention. In the lateral flow assay strip of the present invention shown in FIG. 6, a sample pad 2 is placed on the chromatography medium 4, and then a conjugate release pad 3 is positioned on the sample pad, followed by a wicking pad on the conjugate release pad. (8) is located. The wicking pad, which is set on the binder release pad 3, facilitates dispersion of the liquid sample, increases the solubility of the binder release pad, and also stores the detector and the standard detector that are non-fixedly adsorbed on the binder release pad. It acts to protect it from being deformed or damaged by bacterial contamination.

The wicking pads used in the present invention include, but are not limited to, hydroxylated polyesters. Examples of such hydroxylated polyesters include the product AW14-20T4 from Pall Corporation, USA.

When the liquid sample is loaded onto the wicking pad 8, the detector and the standard detector, which are dispersed while passing through the wicking pad and diffused to the binder release pad 3, are adsorbed on the binder release pad by the liquid sample, It dissolves and reacts with the analytes in the liquid sample to form a binder and moves with the liquid sample to the sample pad 2. The sample pad 2 located beneath the binder release pad acts as an aid to ensure that the liquid sample and the formed binder are sufficient to produce unreacted reagents and analytes that are not reacted in the binder release pad, while the liquid sample and the binder are chromatographed. It buffers to smoothly move to the medium (4). The formed binder is captured by reaction with reagents (capture and standard capture) immobilized on the chromatography medium, moving together as the liquid sample moves to the chromatography medium by capillary action.

Figure 7 shows a lateral flow assay strip as another embodiment according to the present invention. In this lateral induction assay strip, only the sample pad 2 is located on the chromatography medium. The sample pad may serve as a conjugate release pad, since the fluorophore-labeled detector and the standard detector are immobilized thereon. The binder formed on the binder release pad is captured by reaction with reagents (capture and standard capture) immobilized on the chromatographic medium as they move together as the liquid sample moves to the chromatography medium by capillary action.

Alternatively, the sample pad may be free of detectors and standard detectors. In this case, the so-called two-step way is called by mixing the fluorophore-labeled and standard detectors before dropping the liquid sample onto the sample pad and dropping the mixture onto the sample pad. . This two-step approach provides the effect of more than two times the sensitivity compared to when a fluorophore-labeled detector and a standard detector are adsorbed on the sample pad. However, the detector has a disadvantage that it is inconvenient to carry because it must be refrigerated before use. If analyte is present in the liquid sample, a conjugate is formed and the formed binder moves together as the liquid sample moves to the chromatography medium by capillary action, and the reagents (captures and standard capturers) immobilized on the chromatography medium. Is captured in response.

8 is a side view of a lateral flow assay strip as another embodiment according to the present invention. This lateral flow assay strip is a binder pad placed on a chromatography medium and then a sample pad is placed on the binder release pad. After the liquid sample is added to the sample pad, the liquid sample is transferred to the conjugate release pad by diffusion, and if an analyte is present in the test liquid sample, the fluorescently-labeled detector reacts with the analyte to form a conjugate. . The formed binder is captured by reaction with reagents (capture and standard capture) immobilized on the chromatography medium, moving together as the liquid sample moves to the chromatography medium by capillary action.

9 is a side view of a lateral flow assay strip as yet another embodiment according to the present invention. This lateral flow assay strip is identical except that the lateral flow assay strip shown in FIG. 6 and two chromatographic media are stacked. The second chromatography medium 13 is attached to the adhesive 7 on the support 6, and the first chromatography medium 4 extends slightly longer at both ends than the second chromatography medium, so that it is fixed on the second chromatography medium. Both positioned and extended ends are attached to the adhesive on the support 6. The sample pad 2 is positioned at one end and the absorbent pad 5 is located at the other end on the first chromatography medium.

The capturer 11 and the standard capturer 9, 10 are fixed up and down between the interface of the first chromatography medium 4 and the second chromatography medium 13, respectively. At this time, the capturer fixed on the lower surface of the first chromatographic medium may be the same as or different from the standard capturer fixed on the upper surface of the second chromatographic medium. If the same standard capturer is immobilized between two layers of chromatography media, the sensitivity is at least twice as high as if the standard capturer is immobilized on one chromatographic medium. If different standard capturers are immobilized between two layers of chromatography media, the same standard capturer is immobilized between two layers of chromatographic medium or twice as much as standard capturers are immobilized on one chromatographic medium. The advantage is that more analytes can be tested.

In addition, this type of lateral flow assay strip provides the advantage of long term storage and constant sensitivity by not exposing the reaction reagent to the outside.

10 is a side view of a lateral flow assay strip as another embodiment according to the present invention. This lateral flow assay strip is a laminate of a thin, transparent polyester film on top of the lateral flow assay strip shown in FIG. By stacking the polyester film on top of the strip as described above it is possible to maintain a higher sensitivity by suppressing the increase of the background signal (signal) due to the label due to the evaporation of water generated during the test time. In addition, because the strip is exposed to the external environment during testing or storage, the top of the strip can be protected from moisture and contaminants in the air by means of a polyester film laminated on top of the strip, thereby maintaining a constant quality at all times. . In addition, an acrylic adhesive tape that has no effect on sensitivity can be adhered to the top of the strip, which, in addition to the above effect obtained by the polyester film, further solidifies each pad adhering to the support of the strip. give.

11 is a side view of a lateral flow assay strip as yet another embodiment according to the present invention. This lateral flow assay strip is formed by laminating a thin, transparent polyester film or by attaching an acrylic adhesive tape on top of the lateral flow assay strip shown in FIG. 9 to take advantage of the lateral flow assay strip shown in FIGS. 9 and 10. Both are very desirable types that can be provided.

The present invention, in order to quantify the analyte by measuring the intensity of the surface fluorescence, laser, exciter filter, elliptical reflector or spherical mirror, sample control means for emitting surface fluorescence, spatial filter, parallel optical system, fluorescence filter and It consists of a photodetector, and these components focus the light irradiated from the laser onto the sample surface through an excitation filter, and the focusing point is located at the first focal point of the ellipsoidal reflector, and emits light at this first focal point. The scattered light of the fluorescence and the incident light is reflected by the ellipsoidal reflector and focused on the spatial filter as the second focal point of the ellipsoidal mirror, and the light passing through the spatial filter is transformed into parallel light by the parallel optical system and then filtered through the fluorescent filter A laser-induced surface fluorescence detection device for detecting surface fluorescence, characterized in that only pure and fluorescent components are arranged to be incident on the photodetector. The.

Lasers used in the laser-induced surface fluorescence detection device of the present invention typically include a He-Ne laser and a diode laser. Examples of He-Ne lasers include small, portable, precision iodo-stabilized He-Ne lasers developed by the National Research Laboratory of Metrology (NRLM), agency of Industrial Science and Technology (AIST), and Ministry of International Trade and Industry (MITI). (Model NEO-92SI) and Model 05 LYR 173 (Melles Griot, Irvine, Calif.). Diode lasers are more compact and more precise than He-Ne lasers and include infrared or red light diode lasers.

The laser-induced surface fluorescence detection device used in the present invention is composed of a laser, an excitation filter, an elliptical reflector or spherical mirror, a sample control means for emitting surface fluorescence, a spatial filter, a parallel optical system, a fluorescence filter, and a photodetector. The collecting principle of surface fluorescence by the configuration will be described in detail below with reference to FIGS. 12 and 13.

12 shows an apparatus for detecting surface fluorescence by arranging incident light and a sample focused on an ellipsoidal reflector according to the present invention and processing fluorescence emitted after being focused on a second focus into parallel light. The laser emits a wavelength that matches the excitation band maximum of a particular fluorophore. In one embodiment, the laser 21 is a 2 mW He-Ne laser that emits at a wavelength of 594 nm near the absorption maximum of the fluorescent substance label Alexa. For example, a He-Ne laser model 05 LYR 173 (Melles Griot, Irvine, Calif.) May be used as the laser. The light of the laser 21 passes through the incident filter 22 and passes through a hole located at an intermediate point of the elliptical reflector 23 disposed between the incident filter and the sample control means 24. At this time, the laser, the incident filter and the ellipsoidal mirror are preferably arranged side by side so that the light of the laser passes through the hole of the elliptical reflector 23 through the incident filter 22 as appropriate. The light passing through the ellipsoidal reflector is focused to an appropriate size on the surface of the sample fixed to the sample control means 24. The sample may be moved up and down or back and forth (direction perpendicular to the ground) to change the position by the control means so that the light can be optimally focused. This focusing point is located at the first focal point of the elliptical reflector 23. The fluorescence emitted from the first focus and the scattered light of the incident light are reflected by the ellipsoidal reflector 23 and focused on the spatial filter 25 as the second focus of the elliptical mirror. The spatial filter serves to remove noise caused by dust and the like located on the surface of the sample. Light passing through the spatial filter is transformed into parallel light by a collimator 26, and the formed parallel light passes through the fluorescence filter 28, and scattered light is filtered out, and only pure fluorescence is incident on the photodetector 27. do. The fluorescence incident on the photodetector 27 is transmitted to the computer 29 for analysis indicating the signals indicating its fluorescence intensity.

FIG. 13 shows an apparatus for detecting surface fluorescence by focusing fluorescence of a sample focused on incident light at an image point of a spherical mirror using a spherical mirror instead of an elliptical reflector according to the present invention. Therefore, the process of detecting the surface fluorescence of the sample by the apparatus of FIG. 13 is the same as the apparatus of FIG. 12 described above. The light of the laser passes through the incident filter 32 and passes through a hole located at an intermediate point of the spherical mirror 33 disposed between the incident filter and the sample control means 34. At this time, the laser, the incident filter and the ellipsoidal mirror are preferably arranged side by side so that the light of the laser can properly pass through the hole of the spherical mirror 33 through the incident filter 32. The light passing through the ellipsoidal reflector is focused to an appropriate size on the surface of the sample fixed to the sample control means 34. The sample may be moved up and down or back and forth (direction perpendicular to the ground) to change the position by the control means so that the light can be optimally focused. This focusing point is located at the first focal point of the spherical mirror 33. The fluorescence emitted from the first focus and the scattered light of the incident light are reflected by the spherical mirror 33 and focused on the spatial filter 35 as the second focus of the elliptical mirror. The spatial filter serves to remove noise caused by dust and the like located on the surface of the sample. The light passing through the spatial filter is transformed into parallel light by the parallel optical system 36, and the formed parallel light passes through the fluorescent filter 38, and the scattered light is filtered and only pure fluorescent components are incident to the photodetector 37. The fluorescence incident on the photodetector 37 is transmitted to the computer 39 for analysis as a signal indicating its intensity.

In the fluorescence detection device according to the present invention, the surfaces of the samples 24 and 34 are located within the concave portions of the ellipsoidal reflector 23 or the spherical mirror 33, so that almost no fluorescence is emitted to the atmosphere, but the ellipsoidal reflector 23 or the spherical mirror 33 ), It is extremely unlikely to be lost. In addition, since the fluorescence detection device according to the present invention uses parallel optical systems 26 and 36, the fluorescence reflected through the ellipsoidal reflector 23 or the spherical mirror 33 is transformed into parallel light through the equilibrium optical systems 26 and 36. Therefore, it is very unlikely that fluorescence will be lost in the process of entering the photodetectors 27 and 37.

In short, since the laser-induced surface fluorescence detection device according to the present invention uses a reflecting boundary that completely covers the front surface of the sample instead of the lens when collecting the surface fluorescence, more than 97% of the emitted fluorescence is reflected from the reflector toward the detector (incident light Without losses due to incoming holes). Since the double sample and the sample control means shield some of the reflected light, all but the loss due to this reaches the detection optical system. If the diameter of the reflector is 10 cm and the size of the sample control means is 2 x 10 cm, the loss rate by the control means is 20% (this loss includes the reflected fluorescence loss by the incident light inlet). Therefore, in the present invention, the effect of improving the fluorescence collecting efficiency of less than 10% of the existing lens type fluorescent collecting device to 80% or more can be expected. Those skilled in the art will appreciate from the present invention that the smaller the sample control means, the higher the fluorescence collection efficiency.

Fig. 21 shows the construction of a sample control means as one embodiment of the present invention. The sample holder 41 (holder) can be fixed by inserting one side of the sample 44 (mounted on the strip) at the upper part thereof, and a gear wheel at the lower part thereof to be engaged with the transfer means 42. As the conveying means 42 rotates by the driving of the motor, the conveying means 42 can move back and forth along the guide installed in parallel with the conveying means.

In general, the cutoff in the blood of a substance to be measured (microcystine is an environmental substance and is present in the water rather than in the blood) is shown in Table 2 below.

TABLE 2

Marker unit Cutoff CEA ng / ml 〈5 AFP ng / ml 〈15 PSA ng / ml <4 B2M ng / ml <2 NSE ng / ml 〈15 CYFRA21-1 ng / ml 〈3.5 Myoglobin ng / ml 〈70 CK-MB ng / ml 〈3 cTnI ng / ml <One cTnT pg / ml 〈60 BNP pg / ml 〈100 Microcystine pg / ml 〈300

As an additional embodiment according to the invention analytes measurable up to pg / ml are not limited to these but are listed in Table 3 below.

TABLE 3

Analytes unit Cutoff ACTH pg / ml 200-250 Adrenomedullin pg / ml 480 ± 135pg / ml ANP pg / ml 73 pg / ml Angiotensin II pg / ml 21 ± 4pg / ml Calcitonin pg / ml 10 pg / ml CNP pg / ml 7.36 ± 3.0pg / ml Endorphins pg / ml 30 ± 5pg / ml Gastrin pg / ml 26.4 ± 8.4pg / ml Ghrelin pg / ml 87.79 ± 10.27pg / ml NPY pg / ml 70.7 ± 5.9pg / ml Pancreatic polypeptide pg / ml 218 ± 23pg / ml Urotesin II pg / ml 7.70 ± 0.97pg / ml

The laser-induced surface fluorescence detection device according to the invention was found to measure the lowest detection limit concentration of the analyte to pg / ml.

The invention will be explained in more detail through the following examples. However, these examples should be understood as merely illustrative of the present invention and the present invention should not be limited to these examples.

Example 1

Methods of making monoclonal antibodies for use as detectors and capturers

(1) Preparation of culture solution

Dulecco's modified Eagle's media (DMEM) powder was dissolved in 900 ml of DDW and 3.7 g of sodium bicarbonate was added to the resulting solution and the pH was adjusted to 6.9. Sterilize the culture using a 0.45 μm filter and call this solution incomplete DMEM. Bovine Calf Serum and antibiotics penicillin and streptomycin to 10% in 450 ml of incomplete DMEM. ) To make complete DMEM. 5 ml of 100X HT was added to the complete DMEM to prepare a HAT culture by adding HT (hypoxanthin + thymidine) culture solution and 5 ml of 100X HAT (hypoxanthine + aminopterin + thymidine).

(2) antigen preparation and injection

When injected for the first time, the same volume (mainly 0.3 ml) of Complete Freund's Adjuvant was added to purified enzyme protein solution (50 µg), sonicated for 30 seconds, and the solution was added to BALB / c mice. 0.4 ml each was injected. Three weeks after the first injection, the protein solution was mixed with Incomplete Freund's Adjuvant, followed by two or three injections of this injection, followed by cell fusion experiments 3 to 4 at the final immunization. The protein was injected without adjuvant a day ago. The mice used in the experiments use BALB / c, which is 6 to 8 weeks old, regardless of gender.

(3) Preparation of feeder cells

The supporting cells were prepared one or two days before the fusion experiment. The mice were killed at least 10 weeks after birth, and the abdominal skin was carefully removed and 5 ml of 11.6% sugar solution was injected into the abdominal cavity. After 1-2 minutes, more than 3 ml of the injected sugar solution in the abdominal cavity was recovered by centrifugation (2,000 rpm, 3 minutes), and the supporting cells were dissolved in 30 ml of HAT culture medium, and then dropped into 5 96 well plates. Busy. If the mice were small, the abdominal cells were taken from the two, and if they contained red blood cells, they were prepared again.

(4) Preparation of spleen cells

After killing the mice immunized with the antigen, the spleen was taken aseptically, transferred to a culture dish containing 10 ml of incomplete DMEM, the tissue was crushed using fine tweezers, and the spleen cells were released into the culture medium. The cells were transferred to a 15ml tube, and large tissues or unbroken cells were allowed to settle for 2 minutes, and 5ml of the upper portion was collected again. These were collected by centrifugation, the supernatant was removed and dissolved in 3 ml of incomplete DMEM to combine with the prepared myeloma cells. 3 × 10 ⁷ splenocytes were prepared for one cell fusion experiment.

(5) Preparation of myeloma cells

Five days before the cell fusion experiment, the frozen SP2 / O Ag14 cells in the nitrogen tank were taken out and dissolved, and then completely DMEM was added very slowly to recover. After centrifugation, the sunk cells were again dissolved in 10 ml of complete DMEM and passaged at 37 ° C. CO ₂ incubator at two-day intervals. 5 x 10 ⁷ myeloma cells were prepared and used until the cell fusion experiment.

(6) cell fusion

20 ml of incomplete DMEM was added to the cells obtained by mixing the prepared splenocytes and myeloma cells and centrifuging (2,000 rpm, 3 minutes), and the supernatant was completely removed. Subsequent cell fusion reactions are carried out by hand wrapping and maintaining at 37 ° C. The cells that have sunk at the bottom are crushed completely by tapping the tube with a finger, slowly adding 1 ml of 50% PEG solution for 1 minute and shaking for 90 seconds. The fusion process was carried out. Two minutes and thirty seconds after the start of the first drop of PEG (polyethylene glycol) solution, the incomplete DMEM is added and stopped.The first 1 ml of incomplete DMEM is applied for one minute and then to prevent the osmotic shock caused by the PEG solution. A total of 20 ml of incomplete DMEM was slowly added to the tube by 2 ml 1 min and then 3 ml 1 min. Centrifuge the fused cells in this way, add 20 ml of HAT, wash the remaining PEG, and dissolve the resulting cells in 65 ml of HAT. Put two drops onto a 96-well plate containing support cells. C was incubated in a CO ₂ incubator. Three days after cell fusion, three drops of HT culture medium were newly added to each well, and the cells were newly microscopically changed into three HT culture medium at intervals of three days. Usually, hybridoma colonies began to appear after 4 days of fusion and hybridoma screening began around 7 days. 200 μl of the culture supernatant was transferred to a 24-well plate containing 400 μl of PBS, and the cells in the wells that were positive in the ELISA method were transferred to a new 24-well plate containing 1 ml of HT culture and incubated for another 3 to 4 days. 500 μl of this culture medium was placed in a 15 ml tube containing 2 ml of PBS. The cells were positive again by Western blot, transferred to a 6-well plate containing 5 ml of HT culture, grown, and rapidly frozen. Limiting dilution was performed by cloning the cutting cell line.

(7) Freezing method of hybridoma cells

After centrifugation of cells grown in confluent in a 10 ml culture flask, the settled cells were dissolved in 1 ml of Freezing Media (90% calf serum, 10% DMSO), and then placed in a freezing vial and styrofoam. ) In a box was allowed to slowly drop the temperature at -70 ℃. After 2 hours it is quickly transferred to the liquid nitrogen tank for storage, which can be stored almost permanently.

(8) Limited dilution of hybridoma cells

A limiting dilution method was performed to pick out the cells that produced antibodies that responded to one epitope. First, the number of hybridoma cells growing in the log phase is calculated with a Neubauer Cell Counter, and then serial dilutions can contain 15 cells per ml, ie one drop per drop. The cells were allowed to enter and then transferred drop-wise into 96-well plates containing the prepared support cells one or two days ago. The cultures were changed every 3 days and observed with an inverted microscope on day 5 to mark wells with a single colony. Thereafter, the cells were transferred to 24 wells on the 14th, and continued to culture, followed by ELISA, and the hybridoma cells producing the desired antibodies were taken and stored.

(9) Creation of Ascites Fluid

If a large amount of monoclonal antibody is required, BALB / c mice pre-injected with 500 μl of pristane were also injected with 1 × 10 ⁷ revisions of hybridoma cells that produced the desired antibody after 9 days. After 10 to 15 days, anesthetized or lethal mice were moderately enlarged, and ascites was collected using a syringe and centrifuged (4 ° C, 15,000 rpm, 10 minutes) to remove cells and tissues. Divided and stored frozen at -70 ℃ and then separated in IgG using a Protein A column (Protein A column).

(10) Comparison of monoclonal antibodies for each analyte

PSA Free PSA AFP CEA Antigen Cum ^(a) Cum ^(a) Positive ^(b) Human body fluids ^(c) Capture antibody 32c5 (IgG2a) 83c1 (IgG1) 15c3 (IgG2a) 34 (IgG1) Detection antibody 1c1 (IgG2a) 1c1 (IgG2a) 20c4 (IgG1) 17 (IgG2a) Cloth buffer Phosphate Buffer (0.1M, pH 7.4) Tris buffer (0.15M, pH8.0) Borax buffer (0.2M. PH 8.3) Carbonate Buffer (0.5M, pH 9.5) Buffer buffer PBS PBS PBS PBS

(a) from Scripps

(b) obtained from RDI

(c) obtained from Biodesign

Example 2

Protein-Fluorescent Conjugate Preparation

Fluorescent material as a signal source to the mouse monoclonal antibody for the analyte to be measured was polymerized in the following manner and used for the test. The protein to be used for fluorescence binding was purified with high purity of 95% or more, and the concentration showed a good binding ratio of 1 mg / ml or more. In order to facilitate the reaction with the fluorescent material, the purified protein was dialyzed with a buffer solution containing no ammonia and amine ions (0.1 M sodium bicarbonate, pH 8.5) for 12-24 hours in a 4 ° C refrigeration room. Dialysis protein was stored in a freezer below -20 ℃ until use. Alexa 647 fluorescent material (Molecular Probes, USA) as a powder was added directly to the dialyzed protein in the buffer, and the mixture was slowly mixed, followed by reaction using a stirrer for 1-2 hours in a 4 ° C. refrigerator.

Example 3

Purification of Protein-Fluorescent Conjugates

A distribution column packed with Sephadex G-25 was used to remove excess fluorescent material that did not react with the protein-fluorescent polymer and 0.1 M sodium bicarbonate (pH 8.5) was used as the purification buffer. . Purified protein-fluorescent polymer was stored in the refrigerator or freezer below -20 ° C until use.

Example 4

Protein Immobilization of Nitrocellulose Membranes

Proteins were dispensed on nitrocellulose membranes at different concentrations and doses in the form of thin lines using a Biodot Dispenser connected to a syringe pump. After dispensing, the aliquoted protein was immobilized for 2 hours in a dehumidifying apparatus maintained at a temperature of 25 ° C. and a humidity of 35 to 50%. In order to stabilize the protein and prevent nonspecific reactions between the reactants, the dispensed membrane was treated with a stabilizer (mixture of 1% BSA, 0.05% Tween 20, 1% sucrose, 0.1% PVA) and allowed to equilibrate for 5 minutes. Among these stabilizers, BSA can be used by replacing gelatin, Tween 20 with Triton X-100, sucrose with trehalose, PVA (polyvinyl alcohol) with PEG or PVP (polyvinylpyrrolidone), respectively. have. Excess solution of the treated membrane surface was removed and dried at 40 ° C. for 30 minutes. The dried membrane was likewise stored until use in a container maintained at 25 ° C., RH 35-50%.

Example 5

Pretreatment of Sample Pad

The sample pads were pretreated to facilitate the migration of solution components through the nitrocellulose membrane and to maintain the sensitivity of the reaction and to avoid errors in the test results due to nonspecific reactions between the protein-fluorescent polymer and the sample.

Sample pads (2.5 × 30 cm) were allowed to equilibrate for 10 minutes in 1 mL each of the pretreatment solution (20 mM Tris-Cl, 0.1% Triton X-100, 0.05% NaN _3, pH 8.5). When whole blood was used as a sample, other pretreatment solutions (PBS, 10 mM phosphate, 150 mM NaCl, 1% BSA, 0.05% Tween 20, 0.05% NaN ₃ , pH 7.4) were used to prevent erythrocyte hemolysis. Subsequently, excess solution of the sample pad was removed and vacuum dried at a temperature of 50 ° C. to 60 ° C. for 1 hour to prevent deformation of the sample pad by heat. The lyophilization method was chosen to minimize the denaturation of the protein-fluorescent conjugate. The prepared pads were stored until use in storage containers under the same conditions as the membrane above.

Example 6

Combined Release Pad Manufacturing

The protein-fluorescent conjugate, a detector of the substance to be detected, was immobilized on a glass fiber pad to simplify the detection process in one step.

The protein-fluorescent conjugate was diluted in dilution buffer (PBS, 0.1% gelatin, 0.1% Tween 20, pH 7.4) at the ratio of 1/1000, 1/500, and 1/100, respectively. The above mixture is sufficiently wetted with glass fibers, equilibrated for 5 minutes at room temperature, and then dried.However, in order to prevent the mixture from being unevenly redistributed on the surface of the glass fibers and to reduce the amount of the mixture, Instead, microdispensers were dispensed in amounts of 10, 15 and 20ul / cm, respectively. The binder release pad (protein-phosphor conjugate pad) was dried in three ways. First, in consideration of the stability of the protein component was vacuum dried for 6 hours at a temperature of 40 ℃ or less, second, it was dried for 16 hours at room temperature in the dehumidifier. The third takes more time than the first method, but the lyophilization method was chosen to further reduce the possibility of protein inactivation. The prepared conjugate pad was stored until use in a storage container under the same conditions as the above membrane.

Example 7

Protein dispensing on NC (nitrocellulose) membrane

Dilute each protein to be immobilized to 1, 2mg / ml in PBS buffer and dispense onto the NC membrane with 0.88uL / cm line width by using Bio Dot dispenser. Immobilized at 35-50% for 2 hours. The dispensed membrane was then equilibrated for 5 minutes by treatment with a stabilized solution (a mixture of 1% BSA, 0.05% Tween 20, 0.1% PVA) to stabilize the protein and prevent nonspecific reactions between the reactants (BSA in the above stabilizer component gelatin Tween 20 may be replaced by Triton X-100, sucrose by trehalose, and PVA by PEG or PVP respectively). The treated membrane was removed from excess solution on the surface and dried at 40 ° C. for 30 minutes. The membrane to be used for the test was laminated with a sample pad and an absorbent pad and cut into 4 mm widths using a cutter so that the size of the final strip was 4 × 60 mm.

Example 8

Quantification of analytes (single test line)

In the test line section of the NC membrane, capture antibody (1 mg / ml) against PSA (prostate specific antigen) to be analyzed, and rabbit IgG (1 mg / ml, 0.1 mg / ml, 0.05 mg / ml, 0.01 mg) in the standard line. / ml) were each dispensed in an amount of 0.88 μl / cm. Dispensed membranes were immobilized at RH 35-50% for 2 hours. The dispensed membrane was then equilibrated for 5 minutes by treatment with a stabilized solution (mixture of 1% BSA, 0.05% Tween 20, 0.1% PVA) to stabilize the protein and prevent nonspecific reactions between the reactants. The treated membrane was removed from excess solution on the surface and dried at 40 ° C. for 30 minutes. Alexa 647 was fluorescently labeled with the protein to be analyzed by antigen-antibody reaction. In addition, an antibody binding to a protein dispensed at the baseline and antigen-antibody reaction was fluorescently labeled with Alexa 647. The protein-fluorescent conjugate was diluted in a ratio of 1/100 in dilution buffer (PBS, 0.1% gelatin, 0.1% Tween 20, pH 7.4). To this mixture was added 5% trehalose, a stabilizing material, and dispensed in an amount of 20 μl / cm onto the glass fiber surface using a dispenser, and then the protein-fluorescent conjugate pad was lyophilized. The prepared NC membrane and the binder release pad were fixed on the support together with the sample pad and the absorbent pad and assembled in the plastic housing. PSA standard solutions were diluted to dilution buffer (PBST, 10 mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) to prepare 0, 4, 8, 16, 32 ng / ml. The prepared standard solution was reconfirmed using the PSA ELISA kit. In the case of protein dispensed at the standard line, each standard solution prepared above was added dropwise to the sample inlet of the assay kit and injected into the inlet of the laser-induced fluorescence detection device of the present invention 10 minutes later. The device is designed to display the peak of the fluorescence amount of the detector-analyte-capture conjugate stacked on the test and standard lines of the injected assay kit and output the amount on the monitor. The amount of rabbit IgG (0.1 mg / ml) with a peak similar to 8 ng / ml PSA of the analyte was determined as the amount to be dispensed at baseline. After the above standard concentration determination, each PSA standard was added to the assay kit in the same manner. After 10 minutes, the ratio of the amount of fluorescent light deposited on the test line and the standard line was substituted into the equation inferred by polynomial regression. As a result, the amount of fluorescent light in the analyte was converted into a numerical value and output on the screen of the detection device.

Example 9

Quantification of Total / Glass PSAs

Monoclonal antibodies (1 mg / ml) that specifically reacted with total PSA and free PSA were aliquoted and fixed to an amount of 0.88 μl / cm on the NC membrane. Monoclonal antibodies with epitopes other than the immobilized capture antibody were combined with Alexa 647 fluorescent material to prepare antibody-fluorescent conjugates to be used as detectors. The mixture was mixed with PBS buffer containing 5% of trehalose and 1% of gelatin, and diluted to 1/100, soaked in glass fibers in an amount of 50 μl / cm ² , and then lyophilized to prepare an antibody-fluorescent conjugate pad. Was prepared. PSA standard solution was prepared by dilution with dilution buffer (PBST, 10 mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) to 0, 4, 8, 16, 32 ng / ml. The prepared standard solution was reconfirmed using the PSA ELISA kit. Standard concentrations of each concentration were added to the sample input of the test strip and after 15 minutes the intensity of fluorescence in each test zone (total PSA, two zones of free PSA) was measured using the laser induced fluorescence detection device of the present invention. Was measured and quantitatively analyzed. In the case of serum or whole blood, which is a non-standard solution, the concentration was first checked using a PSA ELISA kit, and then the sample was added to a test strip to quantitatively analyze the total PSA and free PSA amounts with a laser-induced fluorescence detection device. The results are shown in FIGS. 19 and 20, respectively.

Example 10

Quantification of AFP, CEA, PSA, CRP (multiple test lines)

α-Peptoprotein (Alpha Feto Protein, AFP, indicator of liver cancer), Carcinoembryonic Antigen (CEA), used for many types of cancer markers or mainly colon cancer), and Metabolite Regulatory Protein (CRP) , A pair of monoclonal antibodies that specifically react with PSA were prepared. Antibodies to be immobilized were sequentially dispensed at 0.8 mm and cm 2 mm from one end of the chromatography medium on the NC membrane. Standard lines were dispensed at a concentration of 100 ug / ml at 2 mm intervals from the top and bottom ends of the test line. Dispensed membranes were immobilized at RH 35-50% for 2 hours. The dispensed membrane was then equilibrated for 5 minutes by treatment with a stabilized solution (mixture of 1% BSA, 0.05% Tween 20, 0.1% PVA) to stabilize the protein and prevent nonspecific reactions between the reactants. The treated membrane was removed from excess solution on the surface and dried at 40 ° C. for 30 minutes. Monoclonal antibodies with antigenic determinants different from the immobilized antibodies above were used as detectors in combination with fluorescent materials. Dilute each polymer with buffer (PBS with 5% trehalose, 1% gelatin, pH 7.4) as in the method for quantifying total / free prostate specific antigen (PSA: 1/100, AFP: 1/200) , CEA: 1/150, CRP: 1/100) 50 μl / cm ² , soaked in glass wool and freeze-dried to prepare an antibody-fluorescent conjugate pad. For each item, the sample whose concentration was determined by the ELISA kit was mixed with dilution buffer (10 mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) and applied to the test strip, followed by laser induced fluorescence of the present invention after 10 minutes. The detection device was quantitatively determined by measuring the intensity of fluorescence in each tester zone. The result is shown in FIG.

Example 11

Preparation of Fluorescently Labeled Antigens or Antibodies

Different kinds of fluorescent substances were bound to antigens and antibodies for comparative analysis. Fluorcecein-isothiocyanate (FITC), rhodamine, and Alexa, Cy3, Cy5 (Molecular Probes, Inc) were tested for binding to antigens and antibodies. The results were good in terms of reproducibility. Subsequent experiments were carried out using Alexa 647 as the basic phosphor. The bound fluorescence-antigen / antibody conjugate showed stable reactivity and could be used without a bleaching effect after a considerable period of time.

Example 12

Determination of Concentration of Protein-Fluorescent Conjugates and Concentration of Fixed Proteins on Nitrocellulose Membranes

Serial dilution experiments were performed to determine the appropriate concentration of detector and capture protein required to detect the material to be measured. The capture protein was fixed on the NC membrane by varying the protein concentration, and then prepared by serial dilution of the protein-fluorescent conjugate. The standard solution of each detection material was mixed with the protein-fluorescer conjugate, mixed and then applied to a test strip, and analyzed by the laser-induced fluorescence detection device of the present invention. The concentration of immobilized protein was 1, 1.2, 1.4, 1.6, 1.8, and 2 mg / ml according to each measurement, and the aliquot was 0.88 ul / cm. When the concentration of the protein was reduced or increased when the concentration of the capture protein was constant, the intensity of the fluorescence signal for the same concentration of the measured substance was decreased or increased. On the contrary, the same result was obtained when the concentration of the protein fluorescent substance conjugate was fixed and the concentration of the capture protein was changed. In both experiments, the nonspecific response increased when the concentration increased beyond some limit. Based on the above results, we determined the concentration of the conditions to minimize the nonspecific reaction between the sample material and the capture and detector while lowering the detection limit of the substance to be measured.

Example 13

Lowest detection limits and linearity of analytes

An appropriate concentration of PSA monoclonal antibody determined in Example 12 was dispensed on the NC membrane in an amount of 0.88 μl / cm, and the antibody-fluorescence conjugate diluted to the value determined in Example 12 was diluted in a strip prepared according to the above method. (PBS containing 5% trehalose, 1% gelatin, pH 7.4), aliquoted into glass fibers in an amount of 20 μl / cm, and freeze-dried to prepare an antibody-fluorescent conjugate pad. PSA standards with varying concentrations from 1 mg / ml to 1 pg / ml were then added to determine the lowest detection limit and linearity range of the assay kit using a fluorescence spectrometer. As shown in FIG. 14, the minimum detection limit of PSA was 10 pg / ml, and the linearity of the PSA assay kit ranged from 10 pg / ml to 1 ug / ml. The lowest detection limits of AFP, CEA and CRP of Example 10 were also able to measure values much lower than the cutoff values required for diagnosis.

Example 14

For comparison with the laser induced fluorescence detection device of the present invention, fluorescence was measured using a conventional laser induced fluorescence detection scanner Scan Life (manufactured by GSI) and the fluorescence intensities were compared with each other.

Analyte PSA was prepared for each concentration, mixed with protein fluorescent material, mixed, and then applied to a strip as in Example 7 to image with the conventional scanner. Lateral flow assay strips prepared with the same methods and conditions were imaged with the laser induced fluorescence detection device of the present invention. The imaged data was digitized using the associated program. The result of this is recorded in FIG. 15. From the recording of FIG. 15, both the laser induced fluorescence detection device according to the present invention and the conventional fluorescence detection scanner showed increased fluorescence intensity according to the concentration of the analyte, but the laser induced fluorescence detection device according to the present invention for the same analyte. It can be seen that the fluorescence intensity measured using is significantly higher than the fluorescence intensity measured using a conventional fluorescence detection scanner.

Example 15

Test according to the position of the internal standard

The strips were made to be positioned forward, backward and forward and backward of the test line to determine the position of the reference line on the strip. Experiments were performed by repeating the prepared strips 10 times for each strip according to the position of the standard line. Experimental method was analyzed by the laser-induced fluorescence detection device of the present invention by applying a standard solution of the detection material prepared for each concentration mixed with the protein-fluorescent material mixture and then mixed in a test strip. The analyzed results were prepared in Table 3 below, and the differences of each strip were compared with the error ranges for the analysis values. The error range of each strip did not fall outside the tolerance range, but it was found that more accurate values could be reproduced when the standard line was positioned forward or forward.

As a result, it was determined that the position of the standard line is located at the front, rear and front and rear of the test line, but it is within the allowable error range, but it is desirable to be located at the front or front and rear for more accurate and reproducible experimental results.

Table 3.

40 pg / ml 400 pg / ml 4 ng / ml All rooms 40.2 ± 4 403.6 ± 12 4.01 ± 0.2 After room 41.3 ± 6 405.2 ± 35 4.09 ± 0.4 Front and back 40.2 ± 3 403.6 ± 11 4.01 ± 0.1

Example 16

Avidin dispensing on NC (nitrocellulose) membrane

The avidin to be immobilized is diluted to 1 or 2 mg / ml in PBS buffer and dispensed onto the NC membrane with 0.88 uL / cm in the amount of 0.88 uL / cm using a Bio Dot Dispenser, followed by RH 35 Immobilized at ˜50% for 2 hours. Subsequently, the aliquoted membrane was treated with a stabilizer (mixture of 1% BSA, 0.05% Tween 20, 0.1% PVA) to equilibrate for 5 minutes (to prevent stabilization of the protein and nonspecific reactions between reactants). Gelatin, Tween 20 can be replaced with Triton X-100, Sucrose with Trehalose and PVA with PEG or PVP respectively). The treated membrane was removed from excess solution on the surface and dried at 40 ° C. for 30 minutes. The membrane to be used for the test was laminated with a sample pad and an absorbent pad and cut into 4 mm widths using a cutter so that the size of the final strip was 4 × 60 mm.

Example 17

Quantification of analytes with avidin-biotin (single test line)

Avidin (1 mg / ml) and rabbit IgG (0.1 mg / ml) were dispensed in the test line region of the NC membrane in an amount of 0.88 μl / cm, respectively. Dispensed membranes were immobilized at RH 35-50% for 2 hours. The dispensed membrane was then equilibrated for 5 minutes by treatment with a stabilizer (mixture of 1% BSA, 0.05% Tween 20, 0.1% PVA) to stabilize the protein and prevent nonspecific reactions between the reactants. The treated membrane was removed from excess solution on the surface and dried at 40 ° C. for 30 minutes. Alexa 647 was fluorescently labeled with the protein to be reacted through the antigen-antibody reaction, and biotin was bound to the protein that was previously captured. In addition, the antibody bound through the antigen-antibody reaction with the protein dispensed at the standard line was fluorescently labeled with Alexa 647. The protein-fluorescent conjugate and protein-biotin conjugate were diluted in a ratio of 1/100 in dilution buffer (PBS, 0.1% gelatin, 0.1% Tween 20, pH 7.4). The prepared NC membrane and the sample pad were fixed on the support together with the absorbent pad, cut to size 4 × 60 mm, and assembled in a plastic housing. PSA standard solution was prepared by dilution with dilution buffer (PBST, 10 mM phosphate, 150 mM NaCl, 0.3% Tween 20, pH 7.4) to 0, 4, 8, 20, 40 ng / ml. The prepared standard solution was reconfirmed using the PSA ELISA kit. Each standard solution prepared above, the protein-fluorescence conjugate and the protein-biotin conjugate, and the protein-fluorescence conjugate which recognizes the substance of the standard line were dropped on the strip included in the package of the present invention, and 10 minutes later, as the inlet of the laser-induced fluorescence detection device. The strip was allowed to deliver. This device displays the peaks of the fluorescence amount of the detector-analyte-capture conjugate stacked on the test line of the strip and the fluorescence amount of the protein-fluorescent conjugate stacked on the standard line, and the fluorescence amount deposited on the test line and standard line. The ratio of was substituted into the equation inferred by polynomial regression, and as a result, the amount of fluorescence of the analyte was converted into a numerical value and displayed on the screen of the detection device. The result is shown in FIG. Line A is the result of quantification using avidin-biotin as described above, and line B is the result of quantification according to the existing method without using avidin-biotin under the same conditions as a control. From the results of FIG. 17, it can be seen that higher sensitivity and higher reproducibility can be obtained than with avidin-biotin.

The lateral flow assay strips according to the present invention are expected to be very useful industrially, particularly medically, as they can quantitate many kinds of analytes simultaneously with high sensitivity.

Claims (27)

Dropping a liquid sample that is expected to contain an analyte at one end of the chromatography medium causes the liquid sample to move through the chromatography medium, and the analyte in the liquid sample removes the chromatography medium from the compartment in which the liquid sample is loaded. And react with labeled detectors adsorbed in compartments spaced at regular intervals in the direction of development, forming a combination of analyte-labeled detectors, which move through the chromatography medium The analyte is captured as a sandwich between the labeled and unlabeled capture by reacting with a capturer that is identical or different from the detector and is unlabeled and immobilized in a test window set at an intermediate position on the chromatography medium. Formed a complex of labeled detector-analyte-unlabeled captures. , In the lateral flow quantitative assay method for determining an analyte in a sample by measuring the amount of complex formed and thus, (a) the labeled detector is labeled with a fluorescent material to react with the analyte in the liquid sample to form a conjugate of the fluorescently labeled detector-analyte; (b) the unlabeled capturer was dispensed in a line to the inspection window on the chromatography medium to react with the conjugate that has migrated along the chromatography medium to form a complex of fluorescently labeled detector-analyte-unlabeled capture; ; (c) on a chromatography medium to which the fluorophore-labeled detector is adsorbed, a standard detector labeled with the same fluorophore as the detector and different from the detector and the trap and reacting with a standard in a liquid sample Unlabeled standard traps adsorbed together and reacted with the fluorescently labeled standard detector forward or backward relative to the inspection window on the chromatographic medium are either fixed as a single line or single in both front and rear. Fixed in lines to form a standard complex of fluorophore labeled standard detector-standards-unlabeled standard capture as the liquid sample moves along the chromatography medium; (d) irradiating light incident on the surface fluorescence medium of the composite and standard composite from the laser and passing through the excitation filter to focus the light reflected therefrom to the first focus of the ellipsoidal or spherical mirror at an appropriate size, The fluorescence emitted from the first focus and the scattered light of the incident light are reflected on the ellipsoidal reflector and focused at the second focus of the ellipsoidal reflector. The focused light is converted back into parallel light by the parallel optical system, and the parallel light is converted by the fluorescent filter. Passing through to filter out scattered light and injecting only pure fluorescent components into the photodetector to determine the amount of analyte by comparing the amount of fluorescence of the complex with respect to the standard amount of fluorescence represented by the standard complex. The method of claim 1 wherein the cutoff value of the analyte is pg / ml. The method of claim 2, wherein the analyte is CEA, PSA, B2M, CYFRA21-1, CK-MB, cTnI, cTNT, BNP, ACTH, adrenomedulin, ANP, angiotensin II, calcitonin, CNP, endorphin, gastrin, NPY , Pancreatic polypeptide, urotensin II and microcystine. The method according to any one of claims 1 to 3, wherein the detectors are four or more types, and accordingly, the same number of catchers as the detector types are divided and fixed in the inspection window with the same number of lines to quantify four or more types of analytes at the same time. Characterized in that the method. The method of claim 1, wherein the standard line to which the unlabeled standard capturer is fixed is located in a single line in front of the inspection window or in a single line in both front and rear. The method of claim 1 wherein the phosphor is selected from the group consisting of Alexa series, Cy3 and Cy5. The method of claim 1, wherein the antibody or antigenic protein used as a capture agent is immobilized by dispensing avidin without dispensing the test line of the test window, and instead attaching biotin to the capture protein so that the biotin binds to avidin and the capture protein is tested. Method automatically detected in the test line of the window. delete delete delete delete delete delete delete delete delete delete (i) a backing, a sample pad adhered to one end of the support and into which a liquid sample is introduced, one end of the sample pad overlapping the end of the sample pad in the direction of the other end of the support, Conjugate releasing pads in which labeled detectors, which react with analytes in the sample to form complexes, are immobilized adsorbed, one end of which overlaps the other end of the labeled release pad toward the other end of the support. By chromatography medium and chromatography, wherein the same or different trappers are immobilized to the support, the sample develops, and the reaction traps in a sandwich and the binder moving apart from the release pad to form a complex. Absorption plaques that absorb moving samples and also absorb and remove labeled unreacted materials An absorption pad; Detectors adsorbed on the conjugated release pad non-fixedly are labeled with a fluorophore and further labeled on the conjugated release pad with the same fluorophore as the labeled fluorescent substance of the detector and react with a standard in a liquid sample. The standard detector is fixedly adsorbed; The capturer is fixed in a line in the inspection window on the chromatography medium; A strip which is different from the detector and the trap and unlabeled standard catcher is fixed in a single line as a standard line in front or rear of the inspection window or in a single line as a standard line in both front and rear of the inspection window; (ii) consisting of a laser, an excitation filter, an ellipsoidal or spherical mirror, a control means for a sample emitting surface fluorescence, a parallel optical system, a fluorescence filter and a photodetector, these components comprising a liquid sample along the chromatographic medium of the strip. The surface fluorescent medium of the complex of fluorophore-labeled detector-analyte-capture formed in the inspection window and the standard complex of fluorophore-labeled standard detector-standard-standard trapper formed at the baseline was transferred from the laser. The incident and reflected light from the excitation filter passes through the ellipsoid to the appropriate size. Focused at the first focal point of the mirror or spherical mirror, the focusing point is located at the first focal point of the ellipsoidal reflector, and the fluorescence emitted from the first focal point and scattered light of the incident light are reflected by the ellipsoidal reflector to be the second focal point of the elliptical reflector A laser-induced surface fluorescence detection device, which is focused on a parallel optical system and is transformed into parallel light and filters scattered light through a fluorescent filter and injects only pure fluorescent components into a photodetector, An integrally configured analyte quantification package such that the amount of fluorescence and the amount of standard fluorescence of the standard complex are measured and relatively compared by the laser-induced surface fluorescence detection device to determine the amount of analyte in the liquid sample. 19. The package of claim 18, wherein the detector and the capturer react with an analyte having a cutoff value of an analyte of pg / ml. The method of claim 18, wherein the analyte is CEA, PSA, B2M, CYFRA21-1, CK-MB, cTnI, cTNT, BNP, ACTH, adrenomedulin, ANP, angiotensin II, calcitonin, CNP, endorphin, gastrin, NPY , Pancreatic polypeptide, urotensin II and microcystine. 21. The method according to any one of claims 18 to 20, wherein the detectors are four or more types, and accordingly, the same number of trappers as the detector types are divided and fixed in the inspection window with the same number of lines so as to quantify four or more types of analytes simultaneously. Can be packaged. 19. The package of claim 18, wherein the standard line to which the unlabeled standard capturer is fixed is located in a single line in front of the inspection window or in a single line in both front and rear. The package of claim 18 wherein the phosphor is selected from the group consisting of Alexa series, Cy3 and Cy5. 19. The package of claim 18, wherein the two chromatography media are stacked and the trap and standard trapper immobilized between the stacked two sides. 19. The package of claim 18 wherein the wicking pad is positioned on the conjugate release pad. The package according to claim 18 or 25, wherein the polyester film or acrylic adhesive tape is integrally laminated on the sample pad, the binder release pad, the chromatographic medium and the absorbent pad. 19. The method according to claim 18, wherein the antibody or antigenic protein used as the capture agent is immobilized by dispensing avidin without dispensing on the test line of the test window, and instead attaching biotin to the capture protein so that the biotin binds to avidin and the capture protein is tested. Packages that are automatically detected on the window's test line.

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