WO2012003617A1 - Immuno-chromatographic detection device and detection method thereof - Google Patents

Abstract

The invention discloses an immuno-chromatography detection device and a detection method thereof. The device comprises a membrane matrix. The membrane matrix is provided with a sample pad, a marker pad carrying a marker which consists of reactive substance and indicative substance, a detection belt coated or chemically cross-linked with the reactive substance and an absorbent pad in turn. The marker is multi-functional polymer formed by the coupling of the reactive substance and the indicative substance on a polymer framework. The detection belt is the reactive substance coated or chemically cross-linked with the membrane matrix or reactive polymer formed by the coupling of the reactive substance on the polymer framework. The detection method is that the sample swims on a membrane and the object to be tested reacts with the marker on the marker pad through the marker pad, then swims to the detection belt to react with the reactive substance under the capillary action, and is developed or checked out through the indicative substance. The invention provides the immuno-chromatography device with high sensitivity and specificity, and the immuno-chromatography detection method with high sensitivity, quickness and reliability.

Description

 Immunochromatographic detection device and detection method thereof

 The invention belongs to the technical field of medical detection, and relates to an immunochromatographic detection device and a detection method thereof. Background technique

Immunochromatography (JC) is a rapid immunoassay developed in the early 1980s. The principle is that by capillary action, the sample moves on the strip fiber membrane, and the substance to be tested is combined with the corresponding ligand preset on the membrane, and the color reaction (or fluorescence or direct use of the coloring marker) is passed in a short time. Intuitive results are obtained within (20mi _n ). Immunochromatography has different names depending on the use of the label, such as enzyme immunochromatography, latex microparticle immunochromatography, colloidal gold immunochromatography, colloidal selenium immunochromatography, colloidal iron immunochromatography, and the like. The most widely used and most mature immunochromatographic technique is colloidal gold immunochromatography. In the domestic market alone, hundreds of colloidal gold immunochromatographic test strips can be easily found, which play a role in early pregnancy testing, infectious disease testing, drug testing, food hygiene testing (such as veterinary drug residues), and genetic testing. An important role. However, the biggest limitation of colloidal gold immunochromatographic detection technology is that the sensitivity and accuracy are difficult to reach the level detected by ELASA. In addition, colloidal gold is prone to agglomeration, and the formed label is unstable, and the labeling of small molecules is poor. Marker specificity is difficult to grasp, and this technical bottleneck greatly limits the further development and application of immunochromatography.

In the immunochromatographic assay, the reaction of the analyte with its reactive substance (substance with specific binding ability such as antibody, nucleic acid or receptor) is not an equilibrium reaction, and the analyte in the sample is composed of a reactive substance. Enrichment is obtained by detecting the band region, so that the efficiency of the membrane chromatography reaction is high. However, the sensitivity of commonly used colloidal gold indicators is low, resulting in low sensitivity of current immunochromatography, which is also colloid. Gold immunochromatography is one of the most important reasons for delaying the replacement of ELISA. Therefore, people's efforts to improve the sensitivity of membrane chromatography technology have never stopped, and the introduction of markers is the most important way to improve the sensitivity of membrane chromatography. The markers used have colloidal gold, colloidal selenium, colloidal iron, latex particles. Liposomes have also been used in recent years, and their aim is to increase the sensitivity of chromatographic detection to a new level. Recently, researchers have used paramagnetic particle labeling, quantum dot labeling and other techniques, or converted the immune response signal into an electronic signal mode to improve sensitivity and achieved good results. However, these new technologies have high requirements for equipment and technology, and there is still a long way to go before practical applications. Summary of the invention

 The technical problem to be solved by the present invention is to provide a high sensitivity and high specificity immunochromatography device for the problems of low sensitivity, poor accuracy, high equipment and technical requirements of the prior art immunodetection device. .

 The technical problem to be further solved by the present invention is to provide a highly sensitive, fast and reliable immunolayering detection method for the problems of low sensitivity, poor accuracy, high equipment and high technical requirements in the prior art.

 The technical solution adopted by the present invention to solve the technical problem thereof is: an immunochromatography detecting device comprising a film substrate, wherein a sample pad and a mark carrying a label composed of a reactive substance and an indicating substance are sequentially disposed on the film substrate. a test strip of a reactive substance, a water-absorbing pad, and a multi-functional polymer formed by coupling a reactive substance and an indicator substance on a polymer skeleton, the test strip A reactive material that is coated or chemically crosslinked on a film substrate or a reactive material formed by coupling a reactive material to a polymer backbone.

The polymer backbone is ruthenium having at least one functional group of -CHO, -NH ₂ , -OH, -COOH. The polymer backbone is dextran, polylysine, polyethylene or polystyrene. The immunochromatographic detection method is: the sample moves on a membrane made of fiber or gel, and the analyte in the sample first reacts with the marker on the marker pad, and then moves to the detection zone under capillary action. The reactive substance reacts, develops color or is detected by an indicator substance; the label is a multifunctional polymer formed by coupling a reactive substance and an indicator substance to a polymer backbone, the detection zone being coated or chemically A reactive material that is crosslinked on the film substrate or a reactive polymer formed by coupling a reactive species to the polymer backbone.

In the immunochromatographic detection method, the polymer skeleton is a polymer having at least one functional group of -CHO, -NH ₂ , -OH, -COOH.

 In the immunochromatographic assay, the polymer backbone may also be dextran, polylysine, polyethylene or polystyrene.

 In the immunochromatographic detection method, a reactive substance or an indicator substance is coupled to a polymer skeleton by a chemical modification or a biological crosslinking method.

 In the immunochromatographic assay, the reactive polymer is coupled to at least one reactive species on the polymer backbone.

 In the immunochromatographic assay method, the multifunctional polymer is coupled to at least one reactive substance and at least one indicator substance on the polymer backbone.

The invention uses a polymer as a skeleton to couple the same function or different functional substances to a specific part of the polymer skeleton by chemical modification or biocrosslinking, thereby imparting multiple biological activities thereto. These substances attached to the polymer backbone can be divided into two according to their functions: one is a reactive substance, and the other is an indicator substance. The reactive substance refers to a substance that performs a specific reaction function such as an antigen, an antibody, an enzyme, a ligand, a receptor, a nucleic acid, or the like. Indicative substances are signal substances such as pigments, fluorescent substances, enzyme quantum dots, etc. that are visible to the naked eye or detectable by the instrument. In membrane chromatography, a polymer with only reactive substances is called reactivity. The polymer is coated or chemically cross-linked to the cellulose membrane to detect the position of the belt; the second is to use a multifunctional polymer carrying both reactive and indicative substances as a marker and added to the label of the membrane chromatography device. On the mat. Such a multifunctional polymer is applied in a membrane chromatography test, in which a substance to be tested in a sample passes through a marker pad to react with a corresponding multifunctional polymer to form a complex, which is pre-coated and reacted with the analyte. When the detection zone is detected, the complex is aggregated in response to the detection zone, and the degree of aggregation can be reflected by enzymatic coloration, fluorescence detection or color judgment. Since the polymer backbone is inert to the membrane and a special treatment to reduce non-specific adsorption is used, the remaining unreacted multifunctional polymer does not adsorb on the membrane and continues to migrate into the water-absorbing material.

 Covalently coupling a reactive substance or an indicator substance to a polymer backbone can change the surface properties and solubility of a reactive substance or an indicator substance, and participate in various water-soluble liquid phase reactions for a substance having poor solubility. Great significance. In terms of test analysis, it acts as a general-purpose reagent to amplify the immune response. The specific principle is: a reactive substance such as an antigen or an antibody is coupled to a polymer backbone by chemically modifying or biologically crosslinking an indicator substance such as a pigment, a fluorescent substance or a horseradish peroxidase (HRP) to form a composite. The complex has multiple reactive groups and can add a large amount of indicator substances according to different needs, thereby increasing the sensitivity and specificity of the immunochromatographic reaction and improving the accuracy of the reaction. At the same time, it retains the rapid, convenient and economic advantages of membrane chromatography technology, and forms a new immunochromatographic detection technology that combines high sensitivity, high specificity, fast and reliable.

In the present invention, a multifunctional polymer is used as a marker instead of colloidal gold, and a highly desirable result is obtained, and the sensitivity is greatly improved without lowering the specificity. The multifunctional polymer not only avoids the disadvantages of poor stability of colloidal gold, difficulty in labeling small molecules and poor labeling specificity, but also improves the stability of the protein (polypeptide), making the detection reagent longer and the detection result more accurate. reliable. The contradiction between the rapid detection and the high sensitivity existing in the modern detection technology is well solved, and the multi-item detection can be simultaneously performed by the multi-marking. In line with modern experimental testing techniques Development direction, with outstanding social and economic benefits. DRAWINGS

 The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:

 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart showing the preparation process of the multifunctional polymer of the embodiment 1 of the present invention.

 Fig. 2 is a view showing the structure of an immunodetection apparatus according to a second embodiment of the present invention. detailed description

 In the first embodiment, the immunochromatographic detection method is: the sample moves on the membrane made of the fiber, and the analyte in the sample first reacts with the label on the membrane, and moves to the detection zone and the reactive substance under capillary action. Reaction, color development; the label is a multifunctional polymer formed by coupling a reactive substance and an indicator substance on a polymer backbone, and the detection strip is coated or chemically crosslinked with a reactive substance coupled to the polymer. A reactive polymer formed on the backbone.

 First, the preparation of multifunctional polymers:

 FIG. 1 is a flow chart showing a process for preparing a multifunctional polymer according to an embodiment of the present invention, in which an antibody is used as a reactive substance, and horseradish peroxidase (HRP) is used as an indicator substance.

1. Preparation of HRP-dextran: First, the glucan as a polymer skeleton was activated under neutral conditions using a sodium periodate method. 10 mg of dextran was weighed, dissolved in 0.01 mol/L of pH 7.2 phosphate buffered saline (PBS), and 0.5 ml of a 0.15 mol/L sodium periodate solution was added thereto, and the mixture was reacted at 25 ° C for 60 minutes. After removing the unreacted sodium periodate from the desalting column, 10 mg of hydrazine (-NH ₂ -NH ₂ -) was added, and the reaction was carried out at 25 ° C for 30 minutes in the dark. Fully dialyzed with 0.01 mol/L phosphate buffer to form aminated dextran; HRP was also activated by sodium periodate method, and 10 mg of HRP was dissolved in 1.0 ml of freshly prepared O.lmol/L NaHCO^§ In the solution, add 0.15mol/L sodium periodate solution 0.5ml, avoiding light at 25 °C After 30 min, the unreacted sodium periodate was removed from the desalting column to form an aldehyde-formed HRP; all the collected colored liquids were added to the above-mentioned aminated dextran, and reacted at 25 ° C for 120 min in the dark, and added to 4.0. Mg/ml sodium borohydride 0.4 ml, 25 ° C in the dark for 60 min, fully dialyzed against 0.01 mol / L phosphate buffer to form HRP-glucan.

 Due to the large molecular weight of dextran, there are many sites for activation, which bring a large amount of amino groups, and since the dextran molecules are long-chain, each skeletal molecule can be coupled with more than 10 horseradish peroxidation. The enzyme (HRP) and HRP-glucan can also be coupled with a fluorescent substance or a dye molecule, and more than 100 fluorescent substances or dye molecules can be coupled. After the coupling is completed, the active group is blocked with ethanolamine.

2. The antibody is digested and reduced by protease: Take 10 mg of antibody, adjust the pH of about 4.2 with 70 mmol/L acetate buffer, add l.Omg of pepsin, and react in a water bath at 37 ° C for 16 hours, using 1.0 mol / The Tris buffer of L was adjusted to pH 8.0, and the Superdex 75 molecular sieve column previously equilibrated with 0.01 mol/L phosphate buffer was used to collect the first elution peak, and concentrated to 5 mg/ml to obtain the antibody fragment F (ab, _2; then, according to the above F (ab,) ₂ per ml of O. lmol / L of dimercapto threitol (DTT) 0.01ml ratio of F (ab') ₂ and DTT, 37 ° C water bath reaction for 90min, After desalting the desalting column, an antibody fragment containing sulfhydryl groups (Fab'-SH) is formed. This process can also activate antibody fragments using a cross-linking agent to form F(ab,) _2- SH.

 3. HRP-dextran is activated by a crosslinking agent: The HRP-dextran prepared in step 1 is concentrated to a concentration of about 5 mg/ml, and a prepared heterobifunctional cross-linking concentration of 10 mg/ml is added per ml. The GMBS (Ν-γ-Maleimidobutyryloxy-oxysuccinimide ester) 0.1ml was incubated at 25 °C for 120 min in the dark. After desalting the desalting column, the first peak was collected as HRP-glucan with maleimide reactive groups. sugar.

 4. The product of step 2, Fab'-SH and the product of step 3, with a maleimide reactive group

HRP-glucan was mixed at a mass ratio of 1:1, protected from light at 25 ° C for 120 min, and glycine 50 mg was added. The reaction was blocked in the dark at 25 ° C for 60 min to block the active group. Finally, the connection of the reactive substance to the polymer is completed to obtain a multifunctional polymer.

The multifunctional polymer thus prepared has a reactive substance on the periphery and an indicator substance on the inner side, which facilitates the participation of the multifunctional polymer in various biological reactions. For the polymer backbone containing -NH ₂ , a bifunctional or trifunctional crosslinker can be directly coupled with an indicator substance and a reactive substance to form a multifunctional polymerization. Preparation of a reactive polymer:

 The antibody is used as a reactive material, polylysine is used as a polymer backbone, and a reactive polymer is prepared by a crosslinking agent method.

 1. Reduction of antibody: The antibody was placed in O. lmol/L pH7.2 PBS, adjusted to a concentration of 10 mg/ml, and added to a final concentration of 10 mmol/L of EDTA; 6 mg of β-mercaptoethylamine was added per ml of antibody. Stir and dissolve, and keep at 37 ° C for 90 min. After Sephadex G-25 or a similar desalting column, the absorption peak at 280 nm was measured to determine the position of the antibody, and the collected antibody was an antibody fragment with -SH.

 2. Activation of polylysine: Polylysine is formulated to a concentration of 10 mg/ml, and a heterobifunctional cross-linking at a concentration of 10 mg/ml is added to each such polylysine. GMBS

(Ν-γ-Maleimidobutyryloxy-oxysuccinimide ester ) 0. lml, 25 ° C in the dark for 120 min, after desalting the desalting column, the first peak was collected as a polylysine with a maleimide reactive group.

 3. Formation of reactive polymer: The antibody fragment with -SH produced in step 1 and the polylysine activated by the product of step 2 are mixed at a mass ratio of 4:1, and reacted at 25 ° C for 120 min in the dark. Excess unbound antibody was removed by Superdex 200 molecular sieve, and the first elution peak was collected as a reactive polymerization term.

Sodium periodate method: a chemical modification method that oxidizes -0H to a CHO and then to a basic strip The method of linking with an NH2 and reducing to form an amide bond is a conventional saccharide-protein linkage technique, and will not be described here.

 Crosslinking agent method: It is a chemical modification method in which a crosslinking agent is used as a bridge to connect different substances and to form stretching arms of different lengths. For the prior art, it will not be described here.

 Biocrosslinking mainly refers to a method of linking two types of substances together in bacteria or cells by means of genetic engineering or the like. Biocrosslinking is a prior art and will not be described here.

 The chemical modification or biological crosslinking method further includes an EDC method in which the carboxylic acid is activated by a carbodiimide (EDC) or a derivative thereof to enable reaction with -NH2, which is a prior art and will not be described herein.

 Coating: The reactive protein or nucleic acid is spotted at the detection zone. After drying, the unbound site is blocked with albumin or casein solution. This process is called coating. The principle of coating is generally a physical adsorption process, and a chemical crosslinking method can also be used.

The immunomembrane chromatography method of the invention can improve the sensitivity from two levels. First, a plurality of reactive substances participating in the reaction are coupled to the polymer backbone, and the relative concentration of the reactive substances is increased, and the membrane chromatography can increase the reactive substances. The chance of contact increases the sensitivity of the reaction. In addition, each polymer backbone can carry more than 10 indicator substances (enzymes, fluorescent substances or more dye molecules), which improves the sensitivity of detection. The membrane in membrane chromatography ensures that the multifunctional polymer can move normally in it, and the polymer backbone (such as dextran) is generally a chromatographic inert substance that does not adsorb to the membrane, ensuring the specificity of the reaction. Coating with a reactive polymer increases the amount of coating, further increasing sensitivity. The polymer skeleton can be coupled with a plurality of reactive substances and indicator substances, and the membrane can be coated with multiple reaction bands, perform various reactions, and display at different positions on the film, so that a plurality of tests can be simultaneously detected. Things. Example 2, Immunochromatography Apparatus: The following is an example of a hepatitis C and HCV infection detection kit. As shown in Figure 2, the hepatitis C and HCV infection detection kit includes a membrane matrix, and the membrane matrix is composed of The polyester film 10 of the portion and the upper chromatographic film 11 are composed of a sample pad 1 and a marker pad 2 carrying a label composed of a reactive substance and an indicator substance, in order from the end of the chromatographic film 11 of the film substrate. Coating tape for coating or chemically cross-linking reactive substances 3, absorbent pad 4

 The marker is a multifunctional polymer formed by using an HCV antigen fragment or an HCV core antibody as a reactive substance and horseradish peroxidase as an indicator substance, which is coupled to a polymer backbone-glucan. The detection bands are multiple, and each of the HCV antigens or HCV core antibodies having different fragments is coated.

 Then, based on the results displayed on the test strip, it is possible to derive the type of fragment to which the antibody is contained in the sample or whether it contains a core antigen. The detection of the HCV antigen antibody kit by multi-functional polymer immunochromatography was carried out and compared with ELISA and colloidal gold reagents. The results are shown in Table 1 below: Table 1: Synchronization of multifunctional polymer immunochromatography Comparison of detection of HCV antigen antibodies with ELISA and colloidal gold

In Table 1, the antigen detection sensitivity of the HCV antigen antibody kit for simultaneous detection of the present invention is 10 pg to the level of ELISA, which is much higher than the detection level of colloidal gold, and the sensitivity of antibody detection is not high, and all three kits are 2.0n _{CU. In} terms of specificity, the present invention simultaneously detects HCV antigen antibody test The kits also reach 100%; for the detection of clinical specimens, compared with the imported kit (antibody detection using the American RIBA kit from Chiron, the antigen detection is the core antigen detection kit of Ortho Corporation of the United States), the coincidence rate of this method Up to 99.2% and 100% respectively; higher than ELISA kit (91.7% for antigen detection, 85% for antibody detection) and colloidal gold kit (79.2% for antigen detection and 81.7% for antibody detection). At the same time, the ELISA kit can only perform a single detection of antigen or antibody in one test. Even if it is detected at the same time, it cannot be detected by fragmentation, while colloidal gold can be detected by partial fragmentation, but it cannot be detected synchronously. In addition, the detection of the HCV antigen antibody kit of the present invention also has obvious technical advantages in terms of detection time and ease of operation.

 In addition, the present invention can also perform combined rapid detection of different viral infections, such as combined rapid detection of blood transfusion testing items such as hepatitis B virus (HBV), hepatitis C virus (HCV), HIV (HIV), and syphilis. HBV surface antibody, HCV core antigen, HIV antigen, and syphilis antigen need to be separately coupled to different polymer backbones, and HBV surface antibodies paired with the label antibody (pro) are coated in different regions on the chromatographic membrane. HCV core antigen, HIV antigen, syphilis antigen, etc. form different detection bands, and when the sample contains the substance to be tested, it will be detected in the corresponding detection zone.

Claims

Rights request 1. An immunochromatographic detection device comprising a membrane substrate, a sample pad disposed on the membrane substrate, a marker pad carrying a label consisting of a reactive substance and an indicator substance, coating or chemical crosslinking reaction A detection tape for a substance, an absorbent pad, characterized in that the marker is a multifunctional polymer formed by coupling a reactive substance and an indicator substance on a polymer skeleton, and the detection zone is coated or chemically crosslinked. The reactive material on the film substrate or the reactive polymer formed by coupling the reactive material to the polymer backbone. The immunochromatographic detection device according to claim 1, wherein the polymer skeleton is a polymer having at least one functional group of -CHO, -NH ₂ , -OH, -COOH.  The immunochromatography detecting apparatus according to claim 1, wherein the polymer skeleton is dextran, polylysine, polyethylene or polystyrene.  4, an immunochromatographic detection method, characterized in that the sample moves on a membrane made of fiber or gel, and the analyte in the sample first reacts with the marker on the marker pad and then swims under capillary action Moving to the detection zone to react with the reactive species, developing color or detecting by the indicator substance; the marker is a multifunctional polymer formed by coupling a reactive substance and an indicator substance to the polymer backbone, the detection zone A reactive polymer formed by coating a reactive substance or a reactive substance on a polymer matrix coated or chemically crosslinked on a film substrate. The immunochromatographic detection method according to claim 4, wherein the polymer skeleton is a polymer having at least one functional group of -CHO, -NH ₂ , -OH, -COOH.  The immunochromatographic detection method according to claim 4, wherein the polymer skeleton is dextran, polylysine, polyethylene or polystyrene. The immunochromatographic detection method according to claim 5 or 6, wherein the reactivity is The substance, indicator substance is coupled to the polymer backbone by chemical modification or biological crosslinking. The immunochromatographic detection method according to claim 7, wherein the reactive polymer has at least one reactive substance coupled to the polymer backbone.  The immunochromatographic detection method according to claim 7, wherein the multifunctional polymer has at least one reactive substance and at least one indicator substance coupled to the polymer backbone.