A method for using a kit for PEA detection

Abstract

The invention provides a kit for detecting PEA and a method for using the kit, comprising PEA protein, anti-PEA antibody, SABC, an enzyme-labeled plate, an enzyme substrate solution, a diluent, a washing solution and a stop solution. The PEA protein is used as a biochemical indicator, the anti-PEA antibody is labeled with biotin as a detection antibody marker, the enzyme-labeled plate coated with the anti-PEA antibody is used as a solid phase carrier, and the SABC is used to amplify the detection signal. The invention uses the biotin-labeled antibody as the detection antibody, adopts a double antibody sandwich method to improve the specificity and accuracy of ELISA detection, and then uses SABC to amplify the detection signal, thereby greatly improving the sensitivity of the detection. Compared with the existing laboratory detection means, the kit is efficient and rapid, has a short operation process, is low in difficulty, and is low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen detection, and in particular to a method for using a kit for PEA detection. Background Art

[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a conditionally pathogenic bacterium that can infect almost all human surface tissues and organs. Because Pseudomonas is widely present in the human body and nature, is insensitive to many antibiotics, and has multiple transmission routes, the hospital infections it causes have become a thorny problem in controlling nosocomial cross-infections.

[0003] The main virulence factor of Pseudomonas is exotoxin A, or PEA, which is an extracellular enzyme produced in the bacteria during bacterial infection. PEA is synthesized as a 71kd precursor protein. After removing the 25 amino acid signal sequence, it is secreted into the supernatant as a 66kd mature protein. It consists of 613 amino acid residues and is divided into three structural functional regions: Region I is the amino acid terminal region, which is related to target cell recognition and can bind to the corresponding surface receptors; Region II is the transmembrane region, which is related to the migration and positioning of the toxin; Region III is the carboxyl terminal region, which has ADP-ribosyltransferase activity and is the active part of the toxin molecule. PEA binds to the surface receptors of target cells and aggregates in the membrane-specialized region through membrane movement. The cell membrane invaginates to form intracellular vesicles, which fuse with lysosomes. After PEA is transferred and positioned in the cytosol of the target cell, it causes cytopathic effects by inhibiting protein synthesis. Studies have confirmed that PEA affects the immune system at non-toxic doses, thereby inhibiting antigen-antibody reactions, and PEA can also inhibit the transformation of lymphocytes in in vitro experiments. Long-term experiments have shown that anti-PEA antibodies have a protective effect in mice infected with Pseudomonas.

[0004] As the main virulence factor of Pseudomonas, PEA has become an important indicator for evaluating Pseudomonas infection. At present, the detection of PEA mainly adopts fluorescence quantitative detection. The RT-PCR equipment used for detection is expensive, the detection process is time-consuming, and the operation is difficult. It is generally only applicable to laboratory research. Therefore, the development of a kit with strong specificity, high sensitivity, easy use, and rapid detection of PEA is of great significance for studying the mechanism of action of Pseudomonas. Summary of the invention

[0005] In view of this, the present invention proposes a method for using a PEA detection kit with rapid detection, strong specificity, high sensitivity and convenience in use.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a kit for PEA detection, including PEA protein, anti-PEA antibody, SABC, ELISA plate, enzyme substrate solution, diluent, washing solution and stop solution, PEA protein is used as a biochemical indicator, anti-PEA antibody is labeled with biotin as a detection antibody marker, the ELISA plate coated with anti-PEA antibody is used as a solid phase carrier, and SABC is used to amplify the detection signal.

[0007] On the basis of the above technical scheme, preferably, the PEA protein is a recombinant protein expressed by Escherichia coli, and its amino acid sequence is GPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAIS.

[0008] On the basis of the above technical scheme, preferably, the anti-PEA antibody is an anti-PEA polyclonal antibody, and its preparation method includes immunizing rabbits with PEA protein to obtain serum containing anti-PEA antibodies, then purifying the serum through a PEA affinity column, and concentrating and dialyzing to obtain purified polyclonal antibodies.

[0009] On the basis of the above technical solution, preferably, the ELISA plate is a 96-well ELISA plate.

[0010] On the basis of the above technical solution, preferably, the enzyme substrate solution is TMB application solution.

[0011] On the basis of the above technical solution, preferably, the diluent is a 10 mmol / L phosphate buffer solution with a pH of 7.2-7.4, containing 1% bovine serum albumin and 0.1% proclin 300.

[0012] On the basis of the above technical solution, preferably, the washing solution is a 10 mol / L phosphate buffer solution with a pH of 7.2-7.4, containing 0.05% Tween-20 and 0.1% proclin 300.

[0013] On the basis of the above technical solution, preferably, the stop solution is 1.0 mol / L H2SO4 solution.

[0014] On the other hand, a method for using a kit for detecting PEA is provided, comprising the following steps:

[0015] S1 dilutes the sample to be tested with a diluent and puts it into the microwells of the ELISA plate to allow the PEA in the sample to be tested to bind to the anti-PEA antibody on the solid phase carrier, and washes away unbound foreign proteins and other substances with a washing solution;

[0016] S2 allows the product obtained in step S1 to bind to the detection antibody marker, and washes away excess biotin-labeled antibody with a washing solution;

[0017] S3. Add SABC to the product obtained in step S2 to form a complex between SABC and the biotin-labeled antibody, and wash away excess SABC with a washing solution;

[0018] S4: adding the enzyme substrate solution to the product obtained in step S3, incubating the reaction at 37° C., and detecting the color intensity after terminating the enzyme reaction with a stop solution.

[0019] The method for using a PEA detection kit of the present invention has the following beneficial effects compared with the prior art:

[0020] (1) The present invention designs a novel kit, uses a biotin-labeled antibody as a detection antibody, adopts a double antibody sandwich method to improve the specificity and accuracy of the detection, and then uses SABC to amplify the detection signal, thereby greatly improving the sensitivity of ELISA detection. Compared with existing laboratory detection methods, it is efficient and rapid, has a short operation process, low difficulty, and low cost.

[0021] (2) The protein used in the present invention is a recombinant protein expressed by Escherichia coli, which has strong specificity, and the antibody obtained by rabbit immunization is a polyclonal antibody. Compared with the monoclonal antibody or the combination of monoclonal antibody and polyclonal antibody commonly used in existing detection methods, the acquisition method is more convenient and simple, and the cost is lower, while ensuring the rapid, accurate and sensitive detection. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] The invention discloses a kit for detecting PEA, comprising PEA protein, anti-PEA antibody, SABC, an enzyme-labeled plate, TMB application solution, 10 mmol / L phosphate buffer solution with a pH value of 7.2-7.4 containing 1% bovine serum albumin and 0.1% proclin 300, 0.25 mol / L phosphate buffer solution with a pH value of 7.2-7.4 containing 0.05% Tween-20 and 0.1% proclin 300, and 1.0 mol / L H2SO4 solution, wherein the PEA protein is used as a biochemical index, the anti-PEA antibody is labeled with biotin as a detection antibody marker, the anti-PEA antibody is coated with a 96-well enzyme-labeled plate as a solid phase carrier, and the SABC is used for amplifying the detection signal.

[0025] Among them, the PEA protein is a recombinant protein expressed by Escherichia coli, and its amino acid sequence is GPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAIS.

[0026] Specifically, the anti-PEA antibody is an anti-PEA polyclonal antibody, and its preparation method includes immunizing rabbits with PEA protein to obtain serum containing anti-PEA antibodies, purifying the serum through a PEA affinity column, and obtaining purified polyclonal antibodies after concentration and dialyzation.

[0027] On the other hand, the method of using the same comprises the following steps:

[0028] S1: dilute the sample to be tested with 10 mol / L phosphate buffer at pH 7.2-7.4 containing 1% bovine serum albumin and 0.1% proclin 300, and then put it into the microwells of the ELISA plate to allow the PEA in the sample to be tested to bind to the anti-PEA antibody on the solid phase carrier, and wash away the unbound foreign proteins and other substances with 10 mol / L phosphate buffer at pH 7.2-7.4 containing 0.05% Tween-20 and 0.1% proclin 300;

[0029] S2 allows the product obtained in step S1 to bind to the detection antibody marker, and washes away excess biotin-labeled antibody with 10 mol / L, pH 7.2-7.4 phosphate buffer;

[0030] S3. Add SABC to the product obtained in step S2 to form a complex between SABC and the biotin-labeled antibody, and wash away excess SABC with a 10 mol / L phosphate buffer solution at pH 7.2-7.4;

[0031] S4: adding TMB application solution to the product obtained in step S3, incubating the reaction at 37° C., and detecting the color intensity after terminating the enzyme reaction with 1.0 mol / L H 2 SO 4 solution.

[0032] It should be noted that ELISA testing, or enzyme-linked immunosorbent assay, refers to a qualitative and quantitative detection method that combines soluble antigens or antibodies with solid phase carriers such as polystyrene and uses the specific binding of antigens and antibodies to conduct immune reactions.

[0033] It should also be noted that the double antibody sandwich method is to bind the anti-PEA antibody to the surface of the enzyme-labeled plate as a solid phase carrier, maintain its immune activity, and connect the anti-PEA antibody to the enzyme to form an enzyme-labeled antibody. This enzyme-labeled antibody retains both its immune activity and the activity of the enzyme. During the determination, the PEA protein and the enzyme-labeled antibody in the sample to be tested undergo an antigen-antibody binding reaction, and then the antigen-antibody complex formed on the solid phase carrier is separated from other substances by a washing method. Finally, the amount of enzyme bound to the solid phase carrier is in a certain proportion to the amount of the substance to be tested in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the substance to be tested in the specimen, so qualitative or quantitative analysis can be performed based on the depth of the color reaction. Since the catalytic efficiency of the enzyme is very high, the reaction effect can be greatly amplified, so that the determination method has a high sensitivity.

[0034] In the present invention, the PEA in the sample to be tested is combined with the anti-PEA antibody on the solid phase carrier in step S1, and the obtained product is combined with the detection antibody marker in step S2, thereby realizing a double antibody sandwich, and then using SABC to amplify the reaction effect, thereby achieving the purpose of improving the detection sensitivity and accuracy.

[0035] Examples 2 and 3 are duplicate hole tests under the same conditions as Example 1. The test results are shown in the following table and Figure 1 As shown. Among them, Figure 1 This is the PEA dose (concentration)-response (absorbance) curve.

[0036] Dose-Response Example 1 Example 2 Example 3 0ng / ml 0.078 0.078 0.078 0.078ng / ml 0.135 0.134 0.135 0.156ng / ml 0.206 0.192 0.200 0.3125ng / ml 0.324 0.322 0.331 0.625ng / ml 0.537 0.538 0.535 1.25ng / ml 0.996 0.954 0.974 2.5ng / ml 1.673 1.655 1.663 5ng / ml 2.262 2.265 2.273

[0037] It can be seen that the detection sensitivity of the present invention is less than 46.9pg / ml, the detection range is 78-5000pg / ml, the linear correlation coefficient of the dose-response curve is greater than 0.990, and it has the advantages of simplicity, sensitivity, accuracy and speed, and provides an effective tool for rapid quantitative detection of PEA.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for using a kit for detecting PEA, characterized in that: The kit for detecting PEA comprises PEA protein, anti-PEA antibody, SABC, ELISA plate, enzyme substrate solution, diluent, washing solution and stop solution, and is characterized in that: the PEA protein is used as a biochemical indicator, the PEA protein is a recombinant protein expressed by Escherichia coli, and its amino acid sequence is GPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAIS; The anti-PEA antibody is labeled with biotin as a detection antibody marker, the ELISA plate coated with the anti-PEA antibody is used as a solid phase carrier, and the anti-PEA antibody is a polyclonal antibody against PEA. The preparation method thereof comprises: immunizing rabbits with PEA protein to obtain serum containing anti-PEA antibodies, purifying the serum through a PEA affinity column, and concentrating and dialyzing to obtain the purified polyclonal antibody; The SABC is used to amplify the detection signal; The ELISA plate is a 96-well ELISA plate; The enzyme substrate solution is TMB application solution; The diluent is a 10 mmol / L phosphate buffer solution with a pH of 7.2-7.4, which contains 1% bovine serum albumin and 0.1% proclin 300; The washing solution is a 10 mol / L phosphate buffer solution with a pH of 7.2-7.4, which contains 0.05% Tween-20 and 0.1% proclin 300; The stop solution is 1.0 mol / L H2SO4 solution; The detection range of the PEA detection kit is 78-5000pg / ml; The following steps are included: S1 dilutes the sample to be tested with a diluent, and then puts it into the microwells of the ELISA plate, so that the PEA in the sample to be tested binds to the anti-PEA antibody on the solid phase carrier, and washes away unbound foreign proteins and other substances with the washing solution; S2 allows the product obtained in step S1 to bind to the detection antibody marker, and uses the washing solution to wash away excess biotin-labeled antibody; S3: adding the SABC to the product obtained in step S2 to form a complex between the SABC and the biotin-labeled antibody, and washing away excess SABC with the washing solution; S4: adding the enzyme substrate solution to the product obtained in step S3, incubating the reaction at 37° C., and detecting the color intensity after terminating the enzyme reaction with a stop solution.

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