Polyelectrolytic internal calibration system of a flow-through assay

Polyelectrolyte internal calibration system for overflow inspection

The present invention continues the U.S. Patent Application No. 10/035014, December 24, 2001.

Various analysis steps and devices are commonly used to detect the presence and/or concentration of analytes present in the test sample using overflow inspection. For example, the immunoassay of the immune system uses a machine in which antibodies are produced in response to the presence of antigens, which are pathogenic or foreign to the organism. These antibodies and antigens, such as an immune response, can bind to one another, so a highly specific reaction machine can be used to detect the presence and concentration of a specific antigen in a biological sample.

These known techniques of immunological methods use immunoreactive tags with detectable complexes, so the analyte can be decomposed and analyzed. For example, a "sandwich format" test generally includes a test sample mixed with an antibody and an analyte. These antibodies are mobile and attached to a tag or probe, such as stained wax, colloidal metal solution, or radioisotope. This mixture is then contacted with a chromatographic analysis material containing a pair of immobilized antibody bands or regions for the analyte. The color layer analysis material usually forms a support plate similar to a measuring ruler. When the analyte and the target antibody are mixed to reach the immobilized antibody on the chromatographic analysis material, binding occurs and the target antibody is bound to a small area in the area. It indicates the presence of the analyte. This technique can be used to include quantitative and semi-quantitative results. Examples of such sandwich form inspection are disclosed in US Patent Nos. 4168146, Grubb et al. and 4,366,241, Tom et al.

An alternative technique is "competitive form" testing. In a "competitive format" test, the label is generally calibrated to the analyte or analyte-analog, which competes for binding with any unlabeled analyte antibodies present in the sample. Competitive testing is generally used to detect analytes such as haptens. Each hapten is monovalent and can only bind to one antibody cell. Examples of competitive immunoassay devices are described in US Patent Nos. 4,235,601, Deutsch et al., 4442204, Liotta, and 5208535, Buechler et al.

Many of these tests rely on calibration to provide educated and meaningful results, especially semi-quantitative and quantitative detection. In particular, external and internal analysis systems are generally used. In an external calibration system, a standard curve generally contains a continuous standard of known analyte quantity, and contains The results of the sample are compared with the standard curve to capture the presence and/or quantity of the analyte in the sample. The external calibration method is quite simple in design and simple to operate. However, it generally interferes with the environment and the degree of change from batch to batch, and is unreliable.

A convenient internal calibration system, in other words, generally uses a film with a calibration zone and a detection zone to fix the analyte capture agent. Unfortunately, a common internal calibration system uses biological capture agents (such as antibodies) in the calibration area. This biological capture agent is expensive, easy to damage, and it is difficult to control the amount of the overall effect. In addition, biological capture agents also tend to reduce time-consuming.

For example, an accurate overflow correction system that needs to exist immediately is controllable, inexpensive, and does not significantly reduce time consumption.

Referring to specific embodiments of the present invention, overflow inspections (such as interlayer, competition, etc.) are revealed to detect the presence and quantity of analytes in the test sample. This test consists of a porous membrane that is paired with a probe containing a special combination and a detectable probe in the liquid flow. For example, in some embodiments, the detectable probes range from luminophores, catalysts, fluorescent substances, chemiluminescent substances, radioactive labels, visible labels, liposomes, and conjugates. In a particular embodiment, the detectable probe contains latex particles.

The porous membrane is also defined as a detection zone containing a capture agent capable of binding the analyte or paired probe. In some specific embodiments, for example, the capture agent can be selected from antigens, haptens, antibodies, and complexes. The detection zone can generate a detection signal to indicate the presence of an analyte.

In addition, to help the detection of the amount of analyte present in the sample, the porous membrane also defines a calibration zone, where the polyelectrolyte is non-dispersively fixed on the porous membrane. The correction zone includes one or more correction zones (such as lines, dots, etc.) containing polyelectrolytes. The polyelectrolyte can be combined with the probe conjugate. The polyelectrolyte used in the calibration zone generally has any required charge. Although not required, the charge of the polyelectrolyte can be selected to be opposite to the charge of the probe, thus helping the formation of ion binding between molecules of relative charge. For example, in a specific embodiment, the polyelectrolyte is negatively charged. In this example, the polyelectrolyte, in some embodiments, includes polylysine, polyethanolamine, epichlorohydrin functional polyamine and/or polyaminoamine, polydipropylene dimethyl aluminum chloride, cationic Choose from cellulose, and the like.

The fixation of the polyelectrolyte in the calibration zone can generally be achieved in a variety of ways. For example, in a specific embodiment, charged polyelectrolyte molecules can form ionic bonds with certain functional groups present in the porous membrane. Similarly, in order to form a more stable and unchanging, polyelectrolyte is sometimes covalently bonded with the functional groups present on the porous membrane. For example, in a specific embodiment, a crosslinkable polyelectrolyte, such as epichlorohydrin functional polyamine and/or polyaminoamine can be crosslinked with the porous membrane.

The signal generated by a calibration zone can then be compared with the detection signal to detect the presence or relative amount of the analyte in the test sample. For example, in some embodiments, the correction signal is visible and can be compared with the detection signal. However, the correction signal can also be compared with the detection signal used by the machine. Such as fluorescent readers, color intensity readers, and similar. If necessary, a calibration curve can be established by plotting the calibration signal with a known amount of analyte. A generated curve is then used to detect unknown amounts of analytes in the test sample.

Referring to another specific embodiment of the present invention, an overflow channel test for detecting the presence and quantity of analytes in a test sample is disclosed. The overflow channel inspection involves a porous membrane that is paired with a probe containing a special combination and a detectable probe between the liquid flow. Upon this contact, the paired probe is sufficient to bind to the analyte in the test sample, so a paired probe/analyte complex and an unbound paired probe are formed. Further, the porous membrane is defined as the detection zone. The capture agent is non-pervasively fixed in the porous membrane in the detection zone. The capture agent can bind to the paired probe/analyte complex to generate a detection signal. Quantitative polyelectrolyte is non-dispersed and fixed on the porous membrane in the calibration zone. The calibration area can generate a calibration signal for comparison with the detection signal, wherein the relative analyte content in the test sample is detected by comparing the detection signal to the calibration signal.

The present invention mentions another specific embodiment, revealing an overflow test for detecting the presence and quantity of an analyte (eg, antigen) in a test sample. The overflow channel inspection involves a porous membrane that is paired with a probe containing a special combination and a detectable probe between the liquid flow. For example, in a specific embodiment, it is a special binding membrane for the analyte. The porous membrane defines a detection zone in which a fixed amount of capture agent is fixed non-pervasively on the porous membrane in the detection zone. The capture agent (eg, antibody) can bind to the analyte (eg, antigen), so the analyte and the paired probe of the test sample compete with a fixed amount of the capture agent. The detection zone can generate a detection signal. Quantitative polyelectrolyte is non-dispersed and fixed on the porous membrane in the calibration zone. The calibration area can generate a calibration signal for comparison with the detection signal, where the relative analyte content in the test sample is determined by the comparison The detection signal wants to correct the signal to detect.

Still referring to another specific embodiment of the present invention, a spillway test for detecting the presence and quantity of an analyte (eg, antigen) in a test sample is disclosed. The test involves a porous membrane that is paired with a probe containing a special conjugate (such as an antibody) and a detectable probe between the liquid flow. Upon this contact, the paired probe is sufficient to bind to the analyte in the test sample, so a paired probe/analyte complex and an unbound paired probe are formed. The porous membrane defines a detection zone in which a fixed amount of capture agent is fixed non-pervasively on the porous membrane in the detection zone. The capture agent (eg, antigen) can be combined with an unbound paired probe, where the detection zone can generate a detection signal. Quantitative polyelectrolyte is non-dispersed and fixed on the porous membrane in the calibration zone. The calibration area can generate a calibration signal for comparison with the detection signal, wherein the relative analyte content in the test sample is detected by comparing the detection signal to the calibration signal.

Other features and viewpoints of the present invention are disclosed in more detail below.

<u style="single">definition</u>

As used herein, "analyte" generally refers to the substance being detected. For example, analytes can include antigenic substances, haptens, antibodies, and conjugates. The analytes include, but are not limited to, toxins, organic substances, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including providing therapeutic purposes as illegal purposes), bacteria, virus particles And metabolites or antibodies to any of the above substances. Some specific examples of analytes include ferritin, creatininase MIB (CK-MB); homodesin; diphenytoin; phenylbarbital; carbamazepine; faecomycin; puromycin; theophylline; Valproic acid; Quinidine; Lutein (LH); Follicle stimulating hormone (FSH); Estradiol; Flavonoids; IgE antibody; Vitamin B2 microglobulin; Glycated hemoglobin (Gly. Hb); Cortisol; Foreign Rehmanniae; N-Acetyl Procainamide (NAPA); Procainamide; German rash virus, such as German rash IgG and German rash IgM; Toxoplasma antibodies, such as Toxo-IgG (Toxo-IgG) ) And Toxo-IgM (Toxo-IgM); Testosterone; Salicylic acid; Acetaminobenzene; Hepatitis B surface antigen (HbsAg); Hepatitis B central antigen antibody, such as anti-Hepatitis B central antigen IgG And IgM (Anti-HBC); Human immunodeficiency virus 1 and 2 (HIV1 and 2); Human T-cell leukemia virus 1 and 2 (HTLV); Hepatitis B e antigen (HbeAg); Type B Hepatitis e antigen antibody (Anti-HBe); Thyroid-stimulating hormone (TSH); Thyroxine (T4); Total thyroxine (Total T3); Free thyroxine (Free T3); Carcinoembryonic antibody (CEA); α-embryo Protein (AFP). Drug abuse and controlled substances include, but tend to restrict, amphetamines; methamphetamines, barbiturates, such as isopentobarbital, cicobarbital, pentobarbital, phenylbarbital Diazide, and barbiturate; benzoin diazoniums, such as chlordiazepoxide, lactam; cannabinoids, such as marijuana and cannabis; cocaine; tanny; ergot diethylamine; methyl ketone; opium, such as heroin , Morphine, codeine, dihydromorphone, dihydrocodeine, methadone, hydroxycodeinone, hydroxydihydromorphine and poppy; phencyclidine; and propaxifen. Other possible analytes are disclosed in U.S. Patent No. 4,366,241, et al.

As used herein, "test sample" is generally referred to as a substance suspected of containing an analyte. The test sample can be used directly, such as containing from the source or pre-processing to modify the characteristics of the sample. The test sample can be obtained from any biological source, such as physiological fluid, containing blood, saliva, tears, cerebrospinal fluid, sweat, urine, breast milk, ascites, rustling, joint fluid, peritoneal fluid, amniotic fluid, or the like. The test sample can be processed before use, such as preparing plasma from blood, diluting mucus, and the like. Treatment methods may include filtration, distillation, concentration, reaction with interferences, and addition of reagents. In addition to physiological fluids, other liquid samples can be used such as water, food products, and performance such as environmental and food inspections. In addition, solid materials suspected of containing analytes can be used like test samples. In some cases, it is beneficial to modify a solid test sample to form a liquid or release an analyte.

<u style="single">A detailed description</u>

Various specific embodiments of the present invention will now be mentioned in detail, and one or more samples will be presented later. Each example is provided by the method of the present invention, but is not limited to the present invention. In fact, it will present these technical skills, and various modifications and changes can be formed by the present invention without departing from the spirit and scope of the present invention. For example, feature descriptions or descriptions are as part of the specific embodiments. Therefore, it is intended that the present invention covers these modifications and changes as within the scope of its patent application.

Generally, the present invention refers to an internal correction system for overflow channel inspection. In particular, the present invention utilizes one or more polyelectrolytes in the calibration zone defined by the tested porous membrane. The polyelectrolyte is constructed to bind the probe and/or the probe combination that flows through the test, thereby generating a correction signal that can be compared with the detection signal. It was discovered that the internal calibration system provides an accurate method to detect the presence of analytes in the test sample, and the materials used in the system are also controllable, inexpensive, and inexpensive. Tend to reduce time-consuming.

Referring to the first to third figures, for example, a specific embodiment of the sandwich-type overflow inspection (20) that can be formed by the present invention will be described in detail below. As shown, the test (20) contains a porous membrane (23) supported by a hard material (not shown). Generally, the porous film (23) can be formed of any of a variety of materials through which the test sample can pass. For example, the materials used to form the porous film (23) include, but are not limited to, natural, synthetic, or naturally occurring synthetic modification materials, such as polysaccharides (cellulose materials such as paper and cellulose derivatives, such as cellulose acetate, And nitrogen cellulose); silica gel; inorganic materials, such as aluminum derivatives, diatomaceous earth, magnesium sulfide, or other inorganic finely dispersed materials-dispersed on a porous polymer basis, and copolymerized with polymers such as chlorinated ethylene and chlorinated ethylene-propylene Materials, and chlorinated ethylene-ethylene acetate copolymer; clothing, naturally occurring (such as cotton) and synthetic (such as nylon or nylon); porous colloids, such as silicone, agar gum, dextran, and gelatin; polymer films, Such as polyacrylamide; and similar. In a particular embodiment, the porous membrane (23) is made of nitrocellulose and/or polyether materials. It must be known that the proper term "nitrocellulose" refers to cellulose nitrate, which can be only cellulose nitrate, or mixed nitrate and other acids, such as aliphatic carboxylic acids with 1 to 7 carbon groups.

Initially detect the analyte (40) in the test sample, a user can directly apply the test sample through a part of the porous membrane (23), which can then move to one or more detection and calibration areas (disclosure Below). Alternatively, the test sample is first applied to the sampling pad, which is in flow contact with the porous film (23). For example, as shown in Figures 1 to 3, the lateral flow inspection (20) may include a sampling pad (21) generally configured to receive samples. Some suitable materials that can be used to form the sampling pad (21) include, but are not limited to, nitrocellulose, cellulose, porous polyethylene pads, and glass fiber filter paper. If necessary, the sample pad (21) can also contain one or more pre-inspection treatment reagents, spread or non-spread adhesion.

In the illustrated embodiment, the test sample moves from the sampling pad (21) to the bonding pad (22) (shown by the arrow (29) in the first figure), which is located at the position where the end of the sampling pad (21) is connected, and the bonding pad (22) Made of materials that the test sample can pass through. For example, in a specific embodiment, the bonding pad (22) is formed of glass fiber.

In addition to simply allowing the test sample to pass through, the bonding pad (22) also performs general functions. For example, in some embodiments, multiple probes (41) (see the second figure) can be released and applied to the bonding pad (twenty two). When included in the binding pad (22), these probes (41) are commercially available to bind the analyte (40), such as the analyte (40) passing through the binding pad (22) through the sampling pad (21). Combined with the analyte (40), the probe (41) can later be used to identify (such as visually, etc.) the presence of the analyte (40) in the detection zone of the test (20).

Any substance that can generate a signal is visually detectable or can be detected by equipment and can be used as a probe (41). A variety of suitable probes can include luminous bodies; emitter labels; visible labels, including colloidal metal and non-metal particles (such as gold), dyed particles, enzymes or substrates, or organic polymer latex particles; liposomes or other containing signals Small sacs that produce matrix; and similar. For example, some enzymes suitable for use as probes are disclosed in U.S. Patent No. 4,275,149, Litman et al., all of which are incorporated herein. Examples of enzyme/substrate probe systems are enzyme base phosphatase and substrate nitrate blue tetrazole-5-bromo-4-chloro-3-indophosphate, or derivatives or similar, or substrate 4-methyl umbellifera Acid-phosphoric acid. In an alternative probe system, the probe can micro-fluorescent substances, there is no enzyme manipulator, and the detection signal needs to be generated. Fluorescent molecules, such as fluorescein yellow, phycoerythrin, rose bengal and its derivatives or similar, are suitable as probes in this reaction. Commercially available examples of this fluorescent material include fluorescent carboxylated microspheres sold by Molecular Probes, Inc. under the trade names "FluoSphere" (red 580/605) and "TransfluoSphere" (543/620), such as " Texas Red, like 5 and 6-carboxytetramethylrhodamine, is also sold by Molecular Probes Inc.

Visually detectable, dyed particles (sometimes referred to as "beads" or "microbeads") can also be used like probes, so the sample can be directly read for the presence or concentration of the analyte without further signals. Producer. In some cases, the particles used in the measurement can also provide signals (such as light absorption) in the region where the particles with different signals stay on the film (23).

The forms of particles used as the probe (41) are also diversified. For example, naturally occurring particles, such as nuclei, bacterioplasms, plastids, cytoplasts, mammalian cells (such as red blood cell hosts), single-celled microorganisms (such as bacteria), polysaccharides (such as agar gum) and the like, can be used. Further, synthetic particles can also be used. For example, in a specific embodiment, synthetic latex particles that can be dyed by a dye can be used like a probe (41). Although any latex particles that can be absorbed or covalently bound to the binding pattern can be used in the present invention, latex particles are generally made of polystyrene, butadiene styrene, styrene acrylic-ethylene terpolymer, and polymethylmethacrylic acid. , Polyethylene methacrylic acid, Styrene-maleic anhydride copolymer, polyethylene acetic acid, polyvinylidine, polyvinylbenzene, polybutylene terephthalic acid, propylene nitrous acid, vinyl chloride-acrylic acid, and similar, or acetaldehyde, carboxyl , Amine, hydroxyl, or hydrazine derivatives. Other suitable particles are disclosed in US Patent Nos. 5,670,381, Joe et al., and 5252459, Tarcha et al., which are incorporated herein. Commercially available suitable dyeing, latex particles include carboxylated latex beads sold by Bang's Laboratory, Inc.

When used, the main diameter of the particle probe (41) is generally diversified as needed depending on factors such as the selected particle form, film pore size, and film composite. For example, in some specific embodiments, the main diameter of the particle probe (41) ranges from 0.01 micrometer to 100 micrometers, and in some specific embodiments, from 0.1 micrometer to 75 micrometers. In a particular embodiment, the particle probe (41) has a diameter of 0.3 microns. In some examples, the pore size of the film (23) is 0.1 to 0.3 microns.

When distributed on the binding pad (22), the probe (41) can directly bind (covalently or non-covalently) with the analyte (40). However, it needs to modify the probe (41) in some ways, so it binds to the analyte (40) more accurately. In some cases, the probe (41) can also be modified with some special binding object (90), which is non-covalent (such as absorption) and/or covalent contact to form a probe conjugate (42).

A special bonding film is generally referred to as a film with a special bonding pair, such as two different molecules, one of which is chemically and/or physically bonded to the second molecule. For example, immunocompetent special binding films include antigens, haptens, and complexes, including those formed by DNA recombination methods or peptide synthesis. The antibody can be a single strain or multiple strains of antibodies, a recombinant protein or a mixture or fragments, the same as a mixture of antibodies and other special binding substances. The detailed preparation of these antibodies and their suitability for use are known technical skills as special binding substances.

Other common special binding pairs include, but are not limited to, biotin and avidin, carbohydrates and glycoproteins, complementary nucleic acid fragments (including probes and the use of DNA hybridization tests to capture nucleic acid fragments to detect target nucleic acid fragments), including by recombination The formed complementary peptide fragments, molecular receptors and actuators, hormones and hormone binding proteins, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, and similar. Further, the molecules contained in the special binding pair are similar to the original special binding substance. For example, analyte fragment derivatives, such as analyte analogs, can be used such as at least one antigen protein commonly found in analytes.

These special combination objects (90) generally can use any variety of techniques to interact with the probe (41) Combine. For example, when using latex particles such as a probe (41), covalently bonding to a special binding object (90) by using carboxyl, amine, acetaldehyde, acetyl bromide, indole, thiol, epoxide and other reactions or bonds The binding functional group, like the residual free radicals and cationic free radicals, completes the reaction through the protein bond. A surface functional group can also be combined like a functional group comonomer because the surface of latex particles can contain a relatively high surface concentration of polar groups. In addition, although latex particle probes are generally functional after synthesis, in some cases, such as polythiol phenol, the particles can be directly covalently bound to semi-white matter without further modification.

Therefore, referring to the second and third figures again, a test sample containing an analyte (40) can initially use the sampling pad (21). From the sampling pad, the test sample can be moved to the binding pad (22), where the analyte (40) binds to the special binding object (90) of the probe conjugate (42) to form a probe conjugate/analyte complex (49). However, because the binding pad (22) is fluidly combined with the porous membrane (23), the probe assembly/analyte complex (49) can migrate from the binding pad (22) to the detection zone (31) present on the porous membrane (23) .

The detection zone (31) may also contain a fixed capture agent (45). Although not required, it is also required that the capture agent (45) can be used from a variety of typical or material types (such as antibodies) as the special binding object (90) to form the probe conjugate (42). These capture agents (45) provide fixed binding sites such as the probe conjugate/analyte complex (49). In some cases, the analyte (40), such as an antibody, antigen, etc., has two binding sites. Upon reaching the detection zone (31), one of these binding positions is occupied by the special binding object (90) of the probe conjugate/analyte complex (49). However, the free binding site of the analyte (40) can bind to the immobilized capture agent (45), and a newly formed trimer (50) probe conjugate (42) indicates the presence of the analyte (40). Therefore, to detect whether the special analyte (40) is present in the test sample, a user can simply analyze the detection area (31).

However, although a detection zone can indicate the presence of an analyte, it is generally difficult to use only the detection zone to detect the relative concentration of the analyte in the test sample. Therefore, referring to the present invention, the test also includes a calibration area, which can be combined with the detection area to detect a specific analyte in the test sample. For example, referring to the first to third figures again, a specific embodiment of the overflow channel inspection (20) including the correction area (32) is described. In this specific embodiment, the correction zone (32) is formed on the porous membrane, and is located at a downstream position of the detection zone (31). The control zone (32) provides a binding agent (47), which can bind any residue The remaining probe (41) and/or the probe conjugate (42) passing through the length of the membrane (23). In particular, starting to contact the test sample, any probe (41) and/or probe knot (42) that does not bind the analyte (40) and complex (49) moves through the detection zone (31). In the detection zone (31), as described above, the complex (49) binds to the capture agent (45) and remains fixed. However, the unbound probe (41) and/or the probe conjugate (42) continue to move through the detection zone (31) and enter the correction zone (32) of the porous membrane (23). In the calibration zone (32), these unbound probes (41) and/or probe conjugates (42) are then combined with the binding agent (47). When fixed to the binding zone (32), the probe (41) and/or the probe binding (42) can be seen by vision or other methods, so the user can compare the signal strength in the detection zone (31) with that in the calibration zone The signal strength in (32).

Referring to specific embodiments of the present invention, the binding agent (47) may include a polyelectrolyte, which may bind the probe (41) and/or the probe conjugate (42). The polyelectrolyte may include a positive or negative valence network, as is generally the case with an electricity valence network formed naturally. For example, some suitable examples of positively charged electrolytes include, but are not limited to, polylysine (available from Sigma-Aldrich Chemical Co., Inc of St. Louis, MO), polyethyleneamine; epichlorohydrin functional poly Amines and/or polyaminoamines, such as polydiethanolamine-co-epichlorohydrin; polydipropylene dimethyl-aluminum chloride; cationic cellulose derivatives, such as cellulose copolymers or cellulose and quaternary water-soluble aluminum Monomer derivatives; and similar. In a specific embodiment, CelQuat<img file="TW587166B_D0001.tif" /> SC-230M or H-100 (available from National Starch &amp; Chemical, Inc.), where the cellulose derivative contains four-stage water-soluble aluminum monomer, can be used. However, there are some suitable examples of polyelectrolytes with negative valence, such as poly(ethylene-co-methacrylic acid, sodium salt) and similar. It must also know the amphoteric polyelectrolyte (if polar and non-polar). For example, some suitable amphoteric polyelectrolytes include, but are not limited to, polyphenyl-bN-methyl-2-vinylridindol) and polyphenylacrylic acid, which are purchased from Polymer Source, Inc. of Dorval, Canada have to.

Although any polyelectrolyte can generally be used, the choice of a particular polyelectrolyte can vary depending on the natural probe/probe combination, porous membrane, and the like. In particular, the scattered electricity prices of polyelectrolytes allow it to be combined with relative electricity prices. Therefore, for example, a polyelectrolyte with a positive charge is generally better assembled and bound to the probe (41) and/or the probe conjugate (42), which is generally negatively charged. Therefore, in some instances, these intermolecular ionic interactions allow the required binding to occur in the correction zone (32). However, although the ion interaction relationship is mainly used to achieve the required combination between the calibration zone (32), it also reveals that the polyelectrolyte can also be used with probes (41) and/or probes with the same charge. Sub-conjugate (42).

Because the polyelectrolyte is designed to combine the probe (41) and/or the probe conjugate (42) B to provide a correction signal, the polyelectrolyte generally required is non-dispersively fixed on the surface of the porous membrane (23). On the contrary, the probe (41) and/or the probe conjugate (42) can be detected immediately by the user through the calibration test. Therefore, the polyelectrolyte can be applied to the porous membrane (23) in some ways, and the polyelectrolyte cannot be dispersed on the basis of the porous membrane (23). In particular, the polyelectrolyte generally forms ions and/or covalently bonds with the functional groups present on the surface of the porous membrane (23), so it remains non-fixed. Although not required, the formation of a covalent bond between the polyelectrolyte and the porous membrane is required to permanently immobilize the electrolyte.

For example, in a specific embodiment, the monomer used to form the polyelectrolyte is first formed in a solution, and then directly applied to the porous membrane (23). Various solutions (such as organic solutions, water, etc.) can be used to form solutions. To be utilized, monomer polymerization initially uses heating, electron beam radiation, free radical polymerization, and the like. In some cases, such as monomer polymerization, it forms a covalent bond with certain porous membrane (23) functional groups, thereby fixing the polyelectrolyte produced. For example, in a specific embodiment, vinylamine monomer can be combined with carboxyl groups present on the surface of some porous membranes (such as nitrocellulose).

In another specific embodiment, the polyelectrolyte can be formed until the porous membrane (23) is used. If necessary, the electrolyte first uses an organic solvent, and something like water is formed. After that, the polyelectrolyte solution is directly applied to the porous membrane (23), and then dried. After drying, the polyelectrolyte can, as described above, form an ionic bond with certain functional groups on the porous membrane. For example, in a specific embodiment, the positively charged polyethanolamine can form an ionic bond with the negatively charged carboxyl groups present on the surface of some porous membranes (such as nitrocellulose).

In addition, the polyelectrolyte can also be connected to the porous membrane (23) using a variety of known techniques. For example, in some embodiments, the epichlorohydrin functional polyamine and/or polyaminoamine can be used as a linkable, positively charged polyelectrolyte. Examples of these materials are disclosed in U.S. Patent Nos. 3700623, keim and 3772076, Keim, 4537657, Keim, all of which are incorporated herein, and are designed by Hercules, Inc., Wilmington, Del. Trademark Kymene^TMsell. Kymene^TM450 and 2064 are epichlorohydrin functional polyamines and/or polyurethane amines containing epoxy and quaternary amines, which can form covalent bonds with the carboxyl groups present in certain porous membranes (such as nitrocellulose), and When acting crosslinks the porous membrane polymer backbone. In some specific embodiments, the temperature range of crosslinking is 50°C to 120°C, and the cross-linking time ranges from 10 to 600 seconds.

Although various techniques of non-dispersive and immobilized polyelectrolyte on the porous membrane (23) are disclosed above, it must be known that any other non-dispersive and immobilized polyelectrolyte composite can be used in the present invention. In fact, the above methods only tend to be exemplary techniques, which can be used in the present invention. For example, in some embodiments, certain complexes can be added to the macroelectrolyte solution, which can inhibit the diffusion of these polyelectrolytes into the porous membrane (23) substrate.

The calibration zone (32) generally can provide any number of different calibration zones, so the user can better detect the concentration of a particular analyte in the test sample. In more specific embodiments, for example, the correction area (32) includes two or more different corrected correction areas (such as lines, points, etc.). For example, in the illustrated embodiment, at least three correction areas (25), (26), and (27) are used to form a line. As shown in the first to third figures, the calibration zone (25), (26) and/or (27) can be located at a position forming a line perpendicular to the direction of flow of the test sample through the test (20).

Similarly, in some specific embodiments, as shown in Fig. 4A, the correction areas (25), (26), and/or (27) may be located at a position forming a line, which is parallel to the test sample passing through Check the direction of flow. In another specific embodiment, as shown in Fig. 4B, the three calibration areas (25a), (26a), and/or (27a) can be located at the positions where the dots are formed, and the parallel test sample passes through the inspection The direction of flow. In some cases, the user can compare the calibration signal with the detection signal in less time, because each calibration area roughly generates a calibration signal.

The calibration zones (25), (26) and/or (27) can be installed on the porous membrane (23) with different amounts of binding agent (47), so each calibration zone (25), (26) and (27) ) The probe (41) and/or the probe conjugate (42) can generate different signal intensities. The total quantity of the binding agent (47) in each calibration zone can be diversified by using calibration zones of different sizes and/or various concentrations or volumes of the binding agent (47) in each calibration zone. Generally, the concentration of the binding agent (47) in the calibration zone ranges from 0.01% to 25% of the weight of the solution.

If necessary, too many probe molecules can be used in the test (20), so each calibration zone (25), (26) and (27) are all reached and the potential signal strength is predicted first. Therefore, the number of probes (41) in the calibration zones (25), (26) and (27) can be pre-determined because the number of binding agents (47) used in the calibration zones (25), (26) and (27) It is the predetermined and known concentration. The comparison can be formed on the intensity of the calibration zone (25), (26) and (27) and the calibration line (24) to calculate the score The amount of analyte (40) present in the test sample. The comparison step takes place, with the help of the reading device or the use of other techniques.

Calibration and test samples can indicate the same situation at the same time, thus providing accurate quantitative results and increased sensitivity. Test (20) can also be used for semi-quantitative detection. In particular, when the multiple calibration zones (25), (26) and (27) provide signal strength ranges, the signal strength of the detection zone (31) can be compared with the signal strengths of the calibration zones (25), (26) and (27) . Based on the intensity range, where the detection zone (31) drops, the general concentration range of the analyte (40) can be detected. If necessary, the signal ratio between the detection zone (31) and the calibration zone (25), (26) and (27) can be plotted as a plane graph with the analyte concentration in the known analyte concentration range to generate a calibration curve, as shown in Figure 5 Shown. To detect the amount of unknown test sample, the signal ratio can then be converted into the analyte concentration of the calibration curve mentioned in the lock. However, when a fluorescent agent is used to detect the amount of analyte 940 in the test sample, a receiver or receiving device can be used to measure the amount of fluorescent agent produced in the detection zone (31) and calibration zone (32), and therefore Make appropriate comparisons to detect the amount of analyte in the test sample.

In addition to the above-mentioned complexes, the spillway inspection (20) can also contain additional complexes. For example, referring to the first to third pictures, the test (20) can also include the wick pad (28). The wick pad (28) receives the liquid that moves through the entire porous membrane (23). As known technical skills, the wick pad (28) can help promote capillary action and liquid flow through the membrane (23).

Although specific embodiments of various inspection devices are described above, it must be understood that the inspection of the present invention generally has any required structure and does not include all the above-mentioned compounds. Furthermore, other compounds known for testing are not specifically mentioned here, and can also be used in the present invention. For example, various inspection structures are described in US Patent Nos. 5395754, Lambotte et al.; 5,670,381, Jou et al., and 6194220, Malick et al., all of which are incorporated herein. In addition, it also knows that the competition test mentioned in the present invention is also formed. The technology and structure of the competition test are known technical skills.

For example, in a specific embodiment, the overflow channel test (20) is disclosed above, which illustrates that the first to third figures can be simply modified to form a competitive test using the probe conjugate (42), which includes a special binding object (90 ) Similarly for the analyte (40), as a result, the analyte (40) and the probe conjugate (42) will compete for the quantified capture agent (45) in the detection zone (31). Generally speaking, because the analyte (40) is not bound, it will quickly pass through the porous membrane and occupy a larger binding site in the detection zone (31). Any unbound probe conjugate (42) will move to the correction area (32). Among them, it can be combined with the binding agent (47). The signal generated in the correction area (32) can be compared with the signal generated in the detection area (31), wherein the relative content of the analyte in the test sample is the inverse ratio of the detection signal intensity and the direct ratio of the correction signal intensity.

Similarly, in another specific embodiment, a competition test can be formed using a capture agent (45), which is also targeted at the analyte (40). Therefore, therefore, in this specific example, the probe conjugate (42) initially binds to the analyte to form a triple complex (49). The unbound probe conjugate (42) and triple complex (49) then move to the detection zone (31). The unbound probe conjugate (42) is combined with the capture agent (45). Any remaining unbound probe conjugate (42) and triple complex (49) will move to the calibration zone (32) where it competes with the quantified binding agent (47). Therefore, the signal generated in the correction area (32) can be compared with the signal generated in the detection area (31), wherein the relative content of the analyte in the test sample is the inverse ratio of the detection signal intensity and the direct ratio of the correction signal intensity.

The present invention is better understood by the following examples.

<b><u style="single">Example one</u></b>

The ability of the calibration zone to effectively utilize multiple polyelectrolytes in the half-test paper sandwich test is proven. Initially, a sample of Milipore SX porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm. The various polyelectrolyte solutions are then dissolved on the membrane sample. The polyelectrolyte solution uses a plastic pipette tip or a dissolving machine to manually dissolve the membrane sample. After using polyethanolamine, the film was dried at 37°C for 1 hour.

The following are the polyelectrolyte solutions tested:<tables><img file="TW587166B_D0002.tif" /></tables><tables><img file="TW587166B_D0003.tif" /></tables>

The laminated film is then cut into small and half test paper shapes. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the half test paper. The other ends of the membrane are embedded with multiple probes and probe conjugate suspensions. In particular, the following probes are tested:<tables><img file="TW587166B_D0004.tif" /></tables>

Half of the test paper is also embedded in the probe conjugate suspension. In particular, the aforementioned probes are combined with anti-CRP Mab, anti-LH Mab and anti-albumin multi-strains using known technology Anti-Pab binding. For example, 100 ml of 0.5 micron fluorescent carboxylated microparticles (purchased by Molecular Probes, Inc.) were initially washed twice with phosphate buffered saline (PBS), and then suspended in 200 ml of PBS for a second time. To the suspension, 5 mg of carbodiether was added and the mixture was mixed for 1 hour. The microspheres were washed twice with boric acid buffer, and then washed again. The microspheres were suspended in 185 ml of boric acid buffer for a second time. 15 ml of α-LH monoclonal antibody (9.7 mg/ml) was then added to the suspension and allowed to react with mixing for 3 hours. Afterwards, 200 ml of 1M ethanolamine aqueous solution was added to the reaction and mixed for 20 minutes. The microspheres were then washed twice with PBS and stored in PBS.

The probe and the probe conjugate inclusion water and 1.6% monolaurate oxyethylene trisitol polymerization (a non-ionic surfactant purchased from Sigma-Aldrich "Tween 20"). The concentration of the produced probe ranges from 0.001 to 5 mg/ml and the concentration of the probe conjugate ranges from 0.2 to 10 mg/ml. After 10 minutes, a separate calibration line for each sample is then noted to detect if the probe/probe combination is visually detectable.

Polylysine, polyethanolamine, polydimethylamine-epichlorohydrin and polydipropylene dimethyl-aluminum chloride show almost complete capture of the probe, and when the capture dose is greater than the probe and the probe conjugate, it is porous Bonded on the membrane. Among the above polyelectrolytes, polylysine and polyethanolamine are the best in terms of capture efficiency (when the number of probes and probe combinations is less than the total dose, a few probes or probe combinations spread across the calibration line); line quality (The shape of the line and the clear edge); and Scattering (the shape of the line remaining after 30 minutes).

In addition, the amphoteric polyisoprene-bN-methyl-2-vinylridinol directly captures the charge and probe complexes. Similarly, the negatively charged polyethylene sodium methacrylate directly captures positively charged latex particles, such as the dyed carboxylated amine-terminated latex particles of Bang's Laboratory, Inc., and their combinations. It also found that a good control/correction line was formed. Interestingly, some polyelectrolytes also capture probes or probe conjugates of the same charge. For example, polyethanolamine captures carboxylation and amine-terminated latex particles and its antibody binds to the membrane to form a control/calibration line. However, trapped particles with the same charge, such as polyelectrolyte, tend to exhibit greater diffusion through the film than trapped particles with a relatively charged polyelectrolyte.

<b><u style="single">Example two</u></b>

The ability of the present invention to control the polyethanolamine in the inner correction zone to correct a half-test paper sandwich test capture is proved. Initially, a sample of Milipore SX porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm. The aqueous solution of polyethanolamine solution was then dissolved on the membrane with a plastic pipette tip at 1.6%, 2%, and 7.4%. After applying polyethanolamine, the film was dried at 37°C for 1 hour.

The laminated film is then cut into small half test paper shapes. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the half test paper. The other ends of the membrane are embedded with a variety of probes and probe binding suspensions as described in Example 1. After immersing in the smearable probe and/or probe binding suspension for 10 minutes, the decomposition correction line of each sample is then observed and detected, if the probe/probe combination is visually detectable.

It was observed that when there were too many blue latex particles (0.3μm, Bang's Laboratory, Inc.), the calibration line formed by 7.4% polyethanolamine solution and the calibration line formed by 2% polyethanolamine was used. In addition, it also detects that when the red fluorescent particles are combined with the anti-α-LH Mab, the calibration line formed by the 7.4% polyethanolamine solution has a stronger intensity than the calibration line formed by the 1.6% polyethanolamine is used.

<b><u style="single">Example three</u></b>

The ability of the polyethanolamine of the present invention to calibrate a half-test paper sandwich test in the inner calibration zone is proved. Initially, a sample of Milipore SX porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm.

A 7.4% polyethanolamine aqueous solution was dissolved on the Milipore SX membrane to form a single calibration line, and the anti-C-reactive protein (anti-CPR) monoclonal antibody (Mab A5804, 1 mg/ml, produced by BiosPacific, Inc. (Purchased) dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. The laminated film is then cut into small half test paper shapes. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane.

One half of the test paper is applied to the control well, which contains Tween 20, the anti-CRP Mab is bound to the blue latex particles (anti-CPR Mab) and water, and the other half of the test paper is applied to the test well and contains C -Reactive protein (CRP), Tween 20, anti-CRP Mab bound to blue latex particles (anti-CPR Mab particles), and water. The mixture in each well moves along the half test paper to the calibration line, the calibration line and the wick pad of the test paper.

The test paper and the mixture are smeared on the test well, the CRP analyte is captured by the anti-CRP Mab particles on the calibration line, and any remaining unbound anti-CRP Mab particles are captured by the polyethanolamine solution on the calibration line. Therefore, after 10 minutes, the blue line appears on the calibration line and the calibration line. Similarly, the test paper and the mixture were smeared on the control well, and all anti-CRP Mab particles were captured on the calibration line. As a result, a blue line only appears on the calibration line.

<b><u style="single">Example four</u></b>

The ability of the polyethanolamine of the present invention to calibrate a full-test paper sandwich test in the inner calibration zone is proven. Initially, two Milipore SX porous membrane samples made of nitrocellulose were laminated on a support plate 30 cm in length. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane, while a cellulose fiber sample pad (Millipore Co.) is attached to the other end of the membrane. A glass fiber bonding pad (Millipore Co.) is also located next to the film of the sample pad.

An aqueous 7.4% polyethanolamine solution was dissolved on the Milipore SX membrane to form a single calibration line. The binding pad is loaded with anti-C-reactive protein (anti-CPR) monoclonal antibody (Mab A5804, 1 mg/ml, purchased from BiosPacific, Inc.) pellets to form a test line. The film was dried at 37°C for 1 hour.

The sample pad of the test paper is then applied to the control well, which only contains phosphate buffered saline (PBS), At the same time, the sample pads of other test papers are applied to the test well, which contains C-reactive protein (CRP), 1.6% Tween 20, and water. The mixture in each well moves along the test paper to the calibration line, the calibration line, and the wick pad of the test paper.

The test paper and the mixture are smeared on the test well, the CRP analyte is captured by the anti-CRP Mab particles on the calibration line, and any remaining unbound anti-CRP Mab particles are captured by the polyethanolamine solution on the calibration line. Therefore, after 10 minutes, the blue line appears on the calibration line and the calibration line. Similarly, the test paper and the mixture were smeared on the control well, and all anti-CRP Mab particles were captured on the calibration line. As a result, a blue line only appears on the calibration line.

<b><u style="single">Example 5</u></b>

The ability of the polyethanolamine of the present invention to calibrate a half-test paper sandwich test in the inner calibration zone is proved. Initially, a sample of Milipore SX porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm.

A 7.4% polyethanolamine aqueous solution was dissolved on the Milipore SX membrane to form a single calibration line, and the anti-β-lutein (anti-β-LH) monoclonal antibody (Mab, 1 mg/ml, supplied by Fitzgerald Industrues Int' I, Inc.) was dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. The laminated film is then cut into small half test paper shapes. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the half test paper.

One half of the test paper is applied to the control well, which contains Tween 20, the anti-α-LH Mab is bound to the blue latex particles (anti-α-LH Mab beads) and water, while the other half of the test paper is applied The test well contains β-lutein (LH), Tween 20, anti-α-lutein (anti-α-LH) Mab bound to blue latex beads (anti-α-LH Mab), and water. The mixture in each well moves along the half test paper to the calibration line, the calibration line and the wick pad of the test paper.

The test paper and the mixture are smeared on the test well, the LH analyte bound to the anti-α-LH Mab particles is captured on the calibration line with the anti-β-LH Mab particles, and any remaining unbound anti-α-LH Mab particles are corrected by the polyethanolamine solution Online capture. Therefore, after 10 minutes, the blue line appears on the calibration line and the calibration line. Similarly, the test paper and the mixture were smeared on the control well, and all anti-α-LH Mab particles were captured on the calibration line. As a result, a blue line only appears on the calibration line.

<b><u style="single">Example 6</u></b>

The ability of the polyethanolamine of the present invention to calibrate a full-test paper sandwich test in the inner correction area is Prove. Initially, a sample of HF 10220 porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm.

A 7.4% ethanolamine aqueous solution was dissolved on the membrane to form a single calibration line, and albuminogen (1 mg/ml, purchased from Biogenesis, Inc.) was dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. The laminated film is then cut into small half test paper shapes. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the half test paper.

One half of the test paper is applied to the control well, which contains 10μl of red fluorescent particles combined with multiple anti-albumin antibodies, while the other half of the test paper is applied to the test well and contains 20 μl of proalbumin (0.2 mg/ml in phosphate buffered saline solution) , 10 ml of red fluorescent particles and multiple anti-albumin antibodies and 40 ml of 2% Tween 20 aqueous solution. The mixture in each well moves along the half test paper to the calibration line, the calibration line and the wick pad of the test paper.

The test paper and the mixture are smeared on the test well, and the proalbumin analyte is captured by the anti-proalbumin multi-strain antibody particles on the calibration line. The albumin binding particles cross the calibration line and are captured on the calibration line. Therefore, after 10 minutes, the red line only appears on the calibration line. Similarly, the test paper and the mixture are smeared on the control well, and the multi-strain anti-albumin antibody particles are first captured on the calibration line, and then some residual strains are captured on the calibration line. As a result, the red line appears on the calibration line and the calibration line.

<b><u style="single">Example 7</u></b>

The ability of the calibration zone of the present invention to calibrate a sandwich test is proven. Initially, a sample of Milipore SX porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm. The aqueous polyethanolamine solution was then dissolved on the membrane (1x, 10x, and 100x diluted 7.4% polyethanolamine solution) to form three separate calibration lines of different concentrations. After using polyethanolamine, the film was dried at 37°C for 1 hour.

A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane. The other ends of the membrane are embedded with multiple probes and probe conjugate suspensions. In particular, the following probes are tested:<tables><img file="TW587166B_D0005.tif" /></tables><tables><img file="TW587166B_D0006.tif" /></tables>

The test also embeds the probe conjugate suspension. In particular, the aforementioned probes are combined with anti-CRP Mab, anti-LH Mab and anti-albumin multi-strains using known technology Anti-Pab binding. For example, 100 ml of 0.5 micron fluorescent carboxylated microparticles (purchased by Molecular Probes, Inc.) were initially washed twice with phosphate buffered saline (PBS), and then suspended in 200 ml of PBS for a second time. To the suspension, 5 mg of carbodiether was added and the mixture was mixed for 1 hour. The microspheres were washed twice with boric acid buffer, and then washed again. The microspheres were suspended in 185 ml of boric acid buffer for a second time. 15 ml of α-LH monoclonal antibody (9.7 mg/ml) was then added to the suspension and allowed to react with mixing for 3 hours. Afterwards, 200 ml of 1M ethanolamine aqueous solution was added to the reaction and mixed for 20 minutes. The microspheres were then washed twice with PBS and stored in PBS.

The probe and the probe conjugate consisted of water and 1.6% oxyethylene tripedol monolaurate polymer (a non-ionic surfactant purchased from Sigma-Aldrich "Tween 20"). The concentration of the produced probe ranges from 0.001 to 5 mg/ml and the concentration of the probe conjugate ranges from 0.2 to 10 mg/ml.

After 5 minutes, the separate calibration line is then noted to detect if the probe/probe combination is visually detectable. The line containing 1x diluent exhibited high signal intensity, while the line containing 100x diluent exhibited lower signal intensity.

<b><u style="single">Example 8</u></b>

The ability of the calibration zone of the present invention to calibrate a half-test paper sandwich test is proven. Initially, a sample of Milipore SX porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm. The 7.4% polyethanolamine solution water sample solution (1x, 10x, and 100x dilution) was then dissolved on the membrane to form three separate calibration lines of different concentrations.

Anti-C-reactive protein (anti-CPR) monoclonal antibody (Mab A5804, 1 mg/ml, purchased from BiosPacific, Inc.) was dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane. The laminated film is then cut into small half test paper shapes. The wick pad is coated with C-reactive protein (CRP), Tween 20, anti-CRP Mab bound to blue latex beads (anti-CPR Mab beads), and water. Thing The qualitative mixture moves along the half test paper to the calibration line, the calibration line, and the wick pad of the test paper.

CRP analytes are captured by anti-CRP Mab beads on the calibration line, while any remaining unbound anti-CRP Mab beads are captured by the calibration line. Therefore, after 5 minutes, a blue line appears on the calibration line, while three blue lines appear in the calibration area. The line containing 1x diluent exhibited high signal intensity, while the line containing 100x diluent exhibited lower signal intensity.

<b><u style="single">Example 9</u></b>

The ability of the calibration zone of the present invention to calibrate a half-test paper sandwich test is proven. Initially, the HF09002 porous membrane sample made of nitrocellulose was laminated on a support plate with a length of 30 cm. 0.14% (Calibration #1), 0.64% (Calibration #2), and 1.4% (Calibration #3) polyethanolamine water sample solution (1x, 10x, and 100x diluted sample) are then dissolved on the membrane to form three strips of different concentrations. Correction line.

Anti-C-reactive protein (anti-CPR) monoclonal antibody (Mab A5804, 1 mg/ml, purchased from BiosPacific, Inc.) was dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane. The laminated film is then cut into small half test paper shapes.

The wick pad is coated with C-reactive protein (CRP), Tween 20, anti-CRP Mab bound to blue latex beads (anti-CPR Mab beads), and water. This test substance also contains different C-reactive protein concentrations. In particular, the solution contains 0 ng, 0.54 ng, 5.4 ng, and 54 ng of CRP.

The substance mixture moves along the half test paper to the calibration line, the calibration line, and the wick pad of the test paper. CRP analytes are captured by anti-CRP Mab beads on the calibration line, while any remaining unbound anti-CRP Mab beads are captured by the calibration line. Therefore, for each sample, a blue line appears on the calibration line, while three blue lines appear in the calibration area. A solution containing 1.4% polyethanolamine exhibited high signal intensity, while a solution containing 0.14% polyethanolamine exhibited lower signal intensity. Based on the analysis, the detected calibration line #1 contains 0.54 ng of CRP, the calibration line #2 contains 5.4 ng of CRP, and the calibration line #3 contains 54 ng of CRP.

Therefore, when testing unknown test samples, the CRP concentration can be detected by comparing the calibration line and the three calibration lines. In particular, when the intensity of the calibration curve is the intensity between the intensities of the detectable calibration line #2 and #3, the CRP concentration is between 5.4 and 54 ng. Similarly, when the intensity of the calibration curve is the intensity between the intensities of the detectable calibration line #1 and #2, the CRP concentration is between 0.54 ng and 5.4 ng. Further, the intensity of the calibration curve is The intensity of the calibration line #1 can be detected, and the CRP concentration is less than 0.54ng. At the same time, the intensity of the calibration line can be detected to have an intensity greater than the intensity of calibration line #3. The CRP concentration is greater than 54ng.

The intensity of the calibration line can also be measured by an instrument, such as an inspection reader. For example, a calibration curve (as shown in the sixth figure) is formed using the line intensities of calibration lines #1 to #3 and their CRP concentration. The mathematical equation generated from the calibration curve can be input into the instrument, which can read the intensity to detect CRP in the test sample.

<b><u style="single">Example ten</u></b>

The ability of the calibration zone of the present invention to calibrate a half-test paper sandwich test is proven. Initially, a sample of SHF 075 porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm. Various concentrations of CelQuat<img file="TW587166B_D0007.tif" /> H-100 is then dissolved on the film to form three separate calibration lines of different concentrations. In particular, the concentration used is 2.5 CelQuat<img file="TW587166B_D0008.tif" /> H-100/million solution (ppm) (calibration line #1), 5ppm (calibration line #2), and 20ppm (calibration line #3).

Anti-β utilizes hormonal (anti-β-LH) monoclonal antibody (Mab, 1 mg/ml, purchased from Fitzgerald Industries Intl. Inc.) to be dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane. The laminated film is then cut into small half test paper shapes.

The wick pad is coated with Tween 20, anti-α-lutein (anti-α-LH) Mab bound to blue latex beads (anti-α-LH Mab beads), and water. The mixture also contains various β-lutein (LH) concentrations. In particular, the concentration test is 0ppm, 20ppm, 100ppm, which conforms to the test concentration including 0ng, 20ng, and 100ngLH.

The substance mixture moves along the half test paper to the calibration line, the calibration line, and the wick pad of the test paper. The LH analyte is captured by anti-α-LH Mab beads on the calibration line, while any remaining unbound anti-α-LH Mab beads are captured by the calibration line. Therefore, for each sample, a blue line appears on the calibration line, while three blue lines appear in the calibration area. Contains 20ppm CelQuat<img file="TW587166B_D0009.tif" /> H-100 solution exhibits high signal intensity and contains 2.5ppm CelQuat<img file="TW587166B_D0010.tif" /> The H-100 solution exhibited lower signal intensity. Based on the analysis, the detected calibration line #1 contains 20ng of LH and calibration line #3 contains 100ng of LH. However, the line intensity can be read using the instrument, and the detection calibration lines #1, #2, and #3 have linear intensities of 1, 2, and 4.

The intensity of the calibration line can also be measured by an instrument, such as an inspection reader. For example, a calibration curve (such as Shown in the seventh figure) Use the line intensities of the calibration lines #1 to #3, and their LH concentration. The mathematical equation generated by the calibration curve can be input into the instrument. The test sample containing unknown concentration is then applied to form the above-mentioned film. Using the instrument, it can detect the detection signal strength of 1.5. As a result, the LH concentration detected in the unknown test sample was 36ng.

<b><u style="single">Example 11</u></b>

The ability of the calibration zone of the present invention to calibrate a half-test paper sandwich test is proven. Initially, a sample of HF 120 porous membrane made of nitrocellulose was laminated on a support plate with a length of 30 cm. Various concentrations of CelQuat<img file="TW587166B_D0011.tif" /> H-100 (a cellulose derivative purchased from National Starch &amp; Chemical, Inc.) was then dissolved on the film to form three separate calibration lines of different concentrations. In particular, the concentration used is 2.5 CelQuat<img file="TW587166B_D0012.tif" /> H-100/million solution (ppm) (calibration line #1), 5ppm (calibration line #2), and 20ppm (calibration line #3).

Albuminogen (1 mg/ml, purchased from Biogenesis, Inc.) was dissolved on the membrane to form a calibration line. The film was dried at 37°C for 1 hour. A cellulose fiber wick pad (Millipore Co.) is attached to one end of the membrane. The laminated film is then cut into small half test paper shapes.

The wick pad is coated with 30ml 2% Tween 20, 10ml red fluorescent particles combined with multiple anti-albumin antibodies, and water on the end of the film. The mixture also contains various concentrations of proalbumin in the phosphate buffered saline solution. Specifically, the test concentrations are 0 mg, 75 mg, and 125 mg.

It can be noticed that the three correction lines switch to different red intensities, where the intensity correction line #3 is the highest and the line #1 is the lowest. In the competition test, the intensity of the calibration curve is in the opposite ratio to the concentration of test albumin. When it is not pro-albumin, the conjugate is captured by the calibration line and three calibration lines. With the increased amount of proalbumin antigen, the calibration line becomes less intense.

The line intensity is then read by a fluorescent reader, and a calibration curve is generated. The results are shown in Table 1.<tables><img file="TW587166B_D0013.tif" /></tables><tables><img file="TW587166B_D0014.tif" /></tables>

The calibration line detects that the signal intensity is 20, 10, and 0 relative to the amount of albumin is 0 mg, 75 mg, and 125 mg. The calibration curve generated from the data is also shown in the eighth figure. Using the calibration curve, the presence and content of albumin of unknown concentration can be detected.

When the present invention discloses these specific embodiments in detail, it will perceive this technical skill, reach the previously unknown, and immediately conceive alternative, altered and equivalent specific embodiments. Therefore, the purpose of the present invention must be determined as additional specific embodiments and anything equivalent.

<p>20. . . sandwich-type flow-through assay</p><p>twenty one. . . sampling pad</p><p>twenty two. . . conjugate pad</p><p>twenty three. . . porous membrane</p><p>twenty four. . . detection line</p><p>25. . . calibration zone</p><p>25a. . . calibration zone</p><p>26. . . calibration zone</p><p>26a. . . calibration zone</p><p>27. . . calibration zone</p><p>27a. . . calibration zone</p><p>28. . . wicking pad</p><p>29. . . arrow arrow</p><p>31. . . detection zone</p><p>32. . . calibration zone</p><p>40. . . analyte</p><p>41. . . probe</p><p>42. . . probe conjugate</p><p>45. . . capture reagent</p><p>47. . . binder</p><p>49. . . complex</p><p>50. . . ternary complex</p><p>90. . . binding member</p>

The first figure is a top view of a specific embodiment of the present invention, showing the overflow channel inspection with three calibration lines in the calibration area;

The second figure is a perspective view of a specific embodiment of the overflow channel of the present invention, showing that the film sheet is applied to the sample pad after the test sample containing the analyte;

The third figure illustrates the lateral inspection shown in the second figure, but through the inspection with the test sample;

The fourth figure is a top view of another specific embodiment of the present invention, wherein the fourth figure A shows the calibration line of parallel analyte flow and the fourth figure B shows the calibration point of parallel analyte flow;

The fifth figure shows a calibration curve used in a specific embodiment of the present invention;

The sixth figure shows the CRP detection calibration curve as disclosed in Example 9;

The seventh figure shows the LH detection calibration curve as disclosed in Example 10;

Figure 8 shows the calibration curve for proalbumin detection as disclosed in Example 11.