CN118191338A - Chemiluminescent immunoassay kit for detecting heat shock protein 70 and preparation method and application thereof - Google Patents
Chemiluminescent immunoassay kit for detecting heat shock protein 70 and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a chemiluminescent immunoassay kit for detecting heat shock protein 70 as well as a preparation method and application thereof. The kit comprises a luminescent plate, a heat shock protein 70 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2, an enzyme-labeled diluent, a 20X concentrated washing solution, a luminescent solution A and a luminescent solution B. The kit for detecting the heat shock protein 70 is a detection kit based on a chemiluminescence immune adsorption method, and has the advantages of high stability, wide linear range, high sensitivity, good repeatability, strong selectivity, high detection speed, low cost, simple and portable instrument, easy operation and the like. Particularly, the sensitivity is improved to 0.039ng/mL, the detection operation time is shortened to 40 minutes, the detection speed is faster, the defects existing in the prior art are effectively overcome, the method has excellent clinical application prospect, and objective basis is provided for clinical diagnosis of hepatocellular carcinoma.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic kits, and particularly relates to a chemiluminescent immunoassay kit for detecting heat shock protein 70 as well as a preparation method and application thereof.
Background
The pathogenesis of hepatocellular carcinoma (HCC) is currently not completely understood, although in recent years there are a number of tumor markers such as: hepatocyte antigen, glypican 3, CD34, CD10, polyclonal carcinoembryonic antigen, arginase 1, glutamine synthetase and the like are used for diagnosis of HCC, but the clinical practice effect is not very ideal. Recent studies have demonstrated that heat shock proteins (heat shock proein, HSP) play an extremely important role in the development of tumors, especially the largest family of HSP70 in the family is often abnormally expressed in tumors caused by various causes, and HSP70 is reported to be involved in HCC apoptosis and immune response, affecting the clinical therapeutic effect of HCC patients. Deregulation of HSP70 expression is a critical causative factor. Therefore, the detection of the content of HSP70 in human body fluid has guiding significance for clinical detection and screening of hepatocellular carcinoma patients.
Currently accepted methods for HSP70 detection include immunoblotting, flow Cytometry (FCM), immunohistochemical staining, and the like. Although these methods have achieved the determination of heat shock protein 70 (HSP 70), there are two important drawbacks: firstly, the sensitivity is low, the detection limit is generally more than 3.0ng/mL, the complex experimental process has long detection time, the general operation time is more than 200 minutes, and the application of heat shock protein 70 (HSP 70) is greatly limited. The national drug administration website is searched, and neither domestic nor import reagents search for heat shock protein 70 (HSP 70) detection kit registration marks. In literature (A novel electrochemical immunosensor based on PG for early screening of depression markers-heat shock protein 70), a novel electrochemical immunosensor based on Porous Graphene (PG) is reported to be developed and used for early screening of a hepatocellular carcinoma biomarker, namely heat shock protein 70 (HSP 70). The detection of heat shock protein 70 (HSP 70) by a chemiluminescent immunoassay method and a detection kit prepared based on the chemiluminescent immunoassay method have not been reported yet.
Therefore, a rapid, effective, simple, convenient and high-sensitivity detection method needs to be urgently established, objective basis is provided for clinical diagnosis, and life health and safety of people are guaranteed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a chemiluminescent immunoassay kit for detecting heat shock protein 70 as well as a preparation method and application thereof, so as to overcome the defects of unstable, low detection sensitivity and long detection time of the conventional chemiluminescent immunoassay kit.
The technical scheme of the invention is as follows:
A chemiluminescent immunoassay kit for detecting heat shock protein 70 comprising: the kit comprises a light-emitting plate, a heat shock protein 70 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2 (Mab2+HRP), an enzyme-labeled diluent, a 20X concentrated washing solution, a light-emitting solution A and a light-emitting solution B.
Further preferably, the luminescent plate is a 96-well or 48-well luminescent plate, and each well of the luminescent plate is coated with heat shock protein 70 monoclonal antibody 1 (Mab 1).
Further preferably, the heat shock protein 70 standard gradient solution comprises 7 heat shock protein 70 standard solutions with concentration gradients of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL, and the heat shock protein 70 standard gradient solution is prepared by a standard buffer solution, wherein the standard buffer solution is a buffer solution containing 5wt% bovine serum albumin and 0.85wt% sodium chloride and having a pH=1.0 mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate.
Further preferably, the sample diluent is formulated as follows: comprises NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、SDS 0.1g、 g casein sodium salt, tritonX-100.1 mL, and Proclin300 mL calculated by 1000mL sterilized pure water.
Further preferably, the amino acid sequence of the heat shock protein 70 monoclonal antibody 1 against an epitope is: DLQQRNSTIQAANLAGLKIL the amino acid sequence of heat shock protein 70 monoclonal antibody 2 against an epitope is: QGGMFLTRAMSGNNKLGGQD; the working titer of the horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2 is 1:10000.
Further preferably, the formulation of the enzyme-labeled diluent is: the sterilized 1000mL pure water contains 8.0g of NaCl, 2.9g of KH 2PO40.2g、Na2HPO4·12H2 O, 9 3g of surfactant S, 5g of casein sodium salt, 10g of BSA and 300mL of Proclin.
Further preferably, the formulation of the 20 x concentrated washing solution is: based on 50mL of sterilized pure water, comprises NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、Tween20 0.5mL.
Further preferably, the luminous solution A is prepared by dissolving carbamide peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare carbamide peroxide stock solution with the concentration of 0.1mol/L, and then gradually diluting the carbamide peroxide stock solution to the working concentration of 4.0X10 -3 mol/L by using the disodium hydrogen phosphate-citric acid buffer solution.
Further preferably, the luminescent solution B is obtained by mixing 3.0X10 -3 mol/L of luminol solution with 3.0X10 -3 mol/L of p-iodophenol solution according to a ratio of 1:1 (v/v).
Further preferably, the luminol solution is prepared into a 0.1mol/L luminol stock solution by using 0.05mol/L, pH of 10.3 carbonate buffer solution, and is placed for three days in a dark place, and then diluted to 3.0X10 -3 mol/L by using 0.1mol/L, pH of 10.3 NaHCO 3 -NaOH buffer solution.
The chemiluminescent immunoassay kit for detecting the heat shock protein 70 also comprises a kit body, self-adhesive, a sealing bag and instructions.
The preparation method of the chemiluminescent immunoassay kit for detecting the heat shock protein 70 comprises the following steps:
1) Preparation of a light-emitting plate:
1.1 Preparing a coating liquid: preparing a phosphate buffer solution with the concentration of 0.01mol/L, and adjusting the pH value to 7.4;
Filtering and sterilizing, and preserving at 4 ℃ for standby;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
Filtering and sterilizing, and preserving at 4 ℃ for standby;
1.3 Preparing a sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
Filtering and sterilizing, and preserving at 4 ℃ for standby;
1.4 Diluting heat shock protein 70 monoclonal antibody 1 (Mab 1) to a working concentration of 10 mug/mL with a coating solution, uniformly mixing, and standing for 15 minutes;
1.5 Taking a light-emitting plate, using a 12-channel gun-discharging spot plate to enable the heat shock protein 70 monoclonal antibody (Mab 1) to reach 100 mu L/hole, covering a cover plate film, placing in a refrigerator with the temperature of 2-8 ℃ and coating for 20-24 hours overnight;
1.6 Taking out the overnight coated luminescent plate, balancing at room temperature for 30min, throwing away the coating liquid, beating to dry with water-absorbing paper, adding 300 mu L/hole of washing liquid each time, standing at room temperature for 1min, washing for 3 times, and beating to dry with water-absorbing paper;
1.7 200 mu L of sealing liquid is added into each hole, and the mixture is sealed for 16 hours at 2-8 ℃ overnight;
1.8 Taking out the sealed coating plate, balancing at room temperature for 30min, throwing off sealing liquid, and drying with absorbent paper; drying in a silica gel dryer, and maintaining humidity below 30% at room temperature for 5 hr;
1.9 Vacuum packaging with aluminum foil bag, marking, and storing at 2-8deg.C for use.
2) Preparation of horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2 (Mab2+HRP)
Marking the heat shock protein 70 monoclonal antibody 2 horseradish peroxidase (Mab2+HRP) according to a sodium periodate method, and placing the marker at 4 ℃ for standby;
3) Preparing a kit solution:
3.1 Preparing enzyme-labeled diluent, wherein the formula of the enzyme-labeled diluent is as follows:
Filtering and sterilizing, and preserving at 4 ℃;
3.2 20 x formulation of concentrated washing solution, the formulation of the 20 x concentrated washing solution is: the sterilized 50mL pure water contains NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL;
3.3 Heat shock protein 70 standard gradient solution is prepared as follows:
Preparing a buffer solution containing 5wt% of bovine serum albumin and 0.85wt% of sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate as a standard buffer solution; preparing 7 heat shock protein 70 standard substance gradient solutions with concentration gradients of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL by using a standard substance buffer solution;
3.4 Preparing a sample diluent, wherein the formula of the sample diluent is as follows: the sterilized 1000mL pure water contains NaCl 8.0g、KH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、SDS 0.1g、.0 g casein sodium salt, triton X-100.1 mL and Proclin300 mL;
3.5 The luminous solution A is carbamide peroxide solution, and the preparation method is as follows:
Dissolving carbamide peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare carbamide peroxide stock solution with the concentration of 0.1mol/L, and gradually diluting to the working concentration of 4.0X10 -3 mol/L by using the disodium hydrogen phosphate-citric acid buffer solution;
3.6 3.0X10 -3 mol/L luminol solution as luminescent solution B and 3.0X10 -3 mol/L p-iodophenol solution are mixed according to the ratio of 1:1 (v/v);
The preparation method of the luminol solution comprises the following steps: preparing 0.1mol/L luminol stock solution by using 0.05mol/L, pH of 10.3 carbonate buffer solution, placing the stock solution for three days in a dark place, and diluting the stock solution to 3.0X10- -3 mol/L by using 0.1mol/L, pH of 10.3 NaHCO 3 -NaOH buffer solution;
in the invention, the antibody titer, the enzyme-labeled antibody titer and the antibody sensitivity are measured according to a chessboard method, so that each point of a standard curve is selected, wherein:
Coating an antibody: the working concentration of the heat shock protein 70 monoclonal antibody 1 (Mab 1) is 10 mug/mL, and the diluent is coating liquid;
Enzyme-labeled heat shock protein 70 monoclonal antibody 2: the working titer of the Mab2+HRP is 1:50000, and the diluent is enzyme-labeled diluent;
The concentrations of each point of the standard curve are 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL respectively, and the diluent is a standard buffer solution.
In the invention, the precision and accuracy of the kit are verified: verifying the precision of the kit by testing a median heat shock protein 70 standard in parallel; the accuracy of the kit is verified in terms of the sample recovery rate of the kit;
In the invention, the sensitivity of the kit is measured: different amounts of heat shock protein 70 were detected with a heat shock protein 70 concentration gradient to determine reagent response sensitivity.
The chemiluminescent immunoassay kit is applied to detection of heat shock protein 70.
The method for detecting the heat shock protein 70 by using the chemiluminescent immunoassay kit comprises the following steps of:
(1) Sample adding: setting a standard substance hole and a sample hole on a light-emitting plate, adding 50 mu L of sample diluent into the standard substance hole according to a concentration gradient, and then adding 50 mu L of heat shock protein 70 standard substance gradient solution; respectively arranging blank holes and sample holes to be detected in the sample holes, adding 100 mu L of sample diluent into the blank holes, adding 50 mu L of sample diluent into the sample holes to be detected, and then adding 50 mu L of sample to be detected; when in sample adding, a sample is added to the bottom of a hole of the plate, the hole wall is not touched as much as possible, and the mixture is gently shaken and uniformly mixed;
(2) Incubation: incubating for 20 minutes at 37 ℃ after membrane sealing by a sealing plate;
(3) Preparing liquid: diluting 20 times of concentrated washing liquid with distilled water for 20 times to obtain washing liquid for standby;
(4) Washing: removing sealing plate membrane, discarding liquid, spin-drying, adding 300 μl of washing liquid into each hole, standing for 30s, discarding, repeating for 5 times, and drying;
(5) Adding enzyme: diluting the Mab2+HRP according to the dilution of 1:50000, wherein the diluent is enzyme-labeled diluent, and 200 mu L of diluted Mab2+HRP solution is added into each hole of the light-emitting plate;
(6) Incubation: incubating for 15 minutes at 37 ℃ after membrane sealing by a sealing plate;
(7) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 20-40 s, discarding, repeating the steps for 5 times, and beating;
(8) And (3) emitting light: adding 50 mu L of luminous liquid A and 50 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing for 4 minutes at 37 ℃ in a dark place;
(9) And (3) measuring: sequentially measuring the luminous intensity of each hole, namely RLU value, at 425nm wavelength by using a blank Kong Diaoling and a full-automatic chemiluminescence analyzer;
(10) Drawing a standard curve on a coordinate paper by taking each concentration of the standard substance as an abscissa and the RLU value as an ordinate, finding out the corresponding concentration of the sample to be tested according to the measured RLU value by the standard curve, and multiplying the corresponding concentration by a dilution factor to obtain the actual concentration of the sample; or calculating a linear regression equation of the standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample.
The beneficial effects are that:
1. The kit for detecting the heat shock protein 70 is a detection kit based on a chemiluminescence immune adsorption method, and has the advantages of high stability, wide linear range, high sensitivity, good repeatability, strong selectivity, high detection speed, low cost, simple and portable instrument, easy operation and the like. Particularly, the sensitivity is improved to 0.039ng/mL, the detection operation time is shortened to 40 minutes, the detection speed is faster, the defects of low sensitivity and low detection speed in the prior art are effectively overcome, and the method has excellent clinical application prospect.
2. The kit can be used for detecting and screening hepatocellular carcinoma patients by detecting the content of the heat shock protein 70 in body fluid, is simple, convenient, quick and effective, and provides objective basis for clinical diagnosis of hepatocellular carcinoma.
3. The kit disclosed by the invention is simple to operate, stable in marker and high in sensitivity, and the defects of complicated and unstable operation steps of the existing detection methods such as enzyme-linked immunosorbent assay, immunoblotting, flow Cytometry (FCM), immunohistochemical staining and the like are completely overcome.
Drawings
FIG. 1 is a standard curve of the chemiluminescent immunoassay kit of example 6.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are merely preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for illustrating the present invention and are not intended to limit the present invention in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
In the following examples, the experimental methods used, not specifically described, were all conventional methods. The reagents used in the examples were all commercially available products.
Example 1: preparation of heat shock protein 70 protein
The corresponding amino acid sequence of heat shock protein 70 is obtained by using the published amino acid sequence DLQQRNSTIQAANLAGLKIL, QGGMFLTRAMSGNNKLGGQD of heat shock protein 70 and using protein expression techniques well known to those skilled in the biological arts.
Constructing a plasmid for expressing the heat shock protein 70, namely a pcDNA3.1-gCD59 expression vector, transfecting CHO cells, screening positive clones, culturing and purifying to obtain the heat shock protein 70 protein.
Example 2: preparation of heat shock protein 70 murine monoclonal antibody Mab1 and heat shock protein 70 murine monoclonal antibody Mab 2.
The obtained heat shock protein 70 protein is immunized into a healthy BALB/c mouse (8 weeks old), and after the mouse generates antibodies, the heat shock protein 70 mouse monoclonal antibody Mab1 and the heat shock protein 70 mouse monoclonal antibody Mab2 are prepared by a conventional myeloma fusion cell technology. The specific process adopts hybridoma antibody preparation technology commonly known to the person skilled in the biological field.
Experiments prove that the amino acid sequence of the prepared heat shock protein 70 antibody Mab1 aiming at the antigen epitope is as follows: DLQQRNSTIQAANLAGLKIL the amino acid sequence of heat shock protein 70 antibody Mab2 against the epitope is: QGGMFLTRAMSGNNKLGGQD.
Example 3
A chemiluminescent immunoassay kit for detecting heat shock protein 70 comprising: the kit comprises a luminescent plate, a heat shock protein 70 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2 (Mab2+HRP), an enzyme-labeled diluent, a 20X concentrated washing solution, a luminescent solution A, a luminescent solution B, a sealing plate film, a sealing bag and a specification;
wherein: the luminous plate is a white opaque polystyrene 96 or 48-hole luminous plate, and each hole of the luminous plate is coated with a heat shock protein 70 monoclonal antibody 1 (Mab 1);
the heat shock protein 70 standard substance gradient solution comprises 6 heat shock protein 70 standard substance solutions with concentration gradients of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL, wherein the heat shock protein 70 standard substance gradient solution is prepared by a standard substance buffer solution, and the standard substance buffer solution is a buffer solution containing 5wt% bovine serum albumin and 0.85wt% sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate;
The formula of the sample diluent is as follows: the sterilized 1000mL pure water contains NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、SDS 0.1g、 g casein sodium salt, tritonX-100.1 mL and Proclin300 mL;
The Mab2+HRP is a product marked by heat shock protein 70 monoclonal antibody 2 horseradish peroxidase (Mab2+HRP) by adopting a sodium periodate method;
The formula of the enzyme-labeled diluent is as follows: the sterilized 1000mL pure water contains 8.0g of NaCl, 2.9g of KH 2PO4 0.2g、Na2HPO4·12H2 O, 9 3g of surfactant S, 5g of casein sodium salt, 10g of BSA and 300 mL of Proclin;
The formula of the 20 x concentrated washing liquid is as follows: the sterilized 50mL pure water contains NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、Tween20 0.5mL;
The luminous solution A is 4.0X10 -3 mol/L carbamide peroxide solution, carbamide peroxide is dissolved in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare 0.1mol/L carbamide oxide stock solution, and then the carbamide peroxide stock solution is gradually diluted to the working concentration of 4.0X10 -3 mol/L by disodium hydrogen phosphate-citric acid buffer solution.
The luminous solution B is obtained by mixing 3.0X10 -3 mol/L luminol solution and 3.0X10 -3 mol/L p-iodophenol solution according to the ratio of 1:1 (v/v);
The luminol solution is prepared into a 0.1mol/L luminol stock solution by using a carbonate buffer solution with the concentration of 0.05mol/L, pH being 10.3, and is placed for three days in a dark place, and then is diluted to 3.0X10 -3 mol/L by using a NaHCO 3 -NaOH buffer solution with the concentration of 0.1mol/L, pH being 10.3.
Each kit comprises 1 block of heat shock protein 70 monoclonal antibody 1 coated luminescent plate, 6 bottles of heat shock protein 70 standard substance gradient solution, sample diluent, mab2+HRP, enzyme-labeled diluent, 20X concentrated washing liquid, luminescent liquid A, luminescent liquid B1 bottle each, one part of instruction book and 3 sealing plate films.
Example 4
The preparation method of the chemiluminescent immunoassay kit for detecting the heat shock protein 70 comprises the following steps:
1) Preparation of a light-emitting plate:
1.1 Preparing a coating liquid: preparing a phosphate buffer solution with the concentration of 0.01mol/L, and adjusting the pH value to 7.4;
Filtering and sterilizing, and preserving at 4 ℃ for standby;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
Filtering and sterilizing, and preserving at 4 ℃ for standby;
1.3 Preparing a sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
Filtering and sterilizing, and preserving at 4 ℃ for standby;
1.4 Diluting shock protein 70 monoclonal antibody 1 (Mab 1) to a working concentration of 10 mug/mL with a coating solution, uniformly mixing, and standing for 15 minutes;
1.5 Taking a luminescent plate, using a 12-channel gun-discharging spot plate to enable the antibody liquid of the Mab1 to reach 100 mu L/hole, covering a cover plate film, placing the luminescent plate in a refrigerator with the temperature of 2-8 ℃ and coating for 20-24 hours overnight;
1.6 Taking out the overnight coated luminescent plate, balancing at room temperature for 30min, throwing away the coating liquid, beating to dry with water-absorbing paper, adding 300 mu L/hole of washing liquid each time, standing at room temperature for 1min, washing for 3 times, and beating to dry with water-absorbing paper;
1.7 200 mu L of sealing liquid is added into each hole, and the mixture is sealed for 16 hours at 2-8 ℃ overnight;
1.8 Taking out the sealed coating plate, balancing at room temperature for 30min, throwing off sealing liquid, and drying with absorbent paper; drying in a silica gel dryer, and maintaining humidity below 30% at room temperature for 5 hr;
1.9 Vacuum packaging with aluminum foil bag, marking, and storing at 2-8deg.C for use.
2) Preparation of horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2
Marking the heat shock protein 70 monoclonal antibody 2 horseradish peroxidase (Mab2+HRP) according to a sodium periodate method, and placing the marker at 4 ℃ for standby;
3) Preparing a kit solution:
3.1 Preparing enzyme-labeled diluent, wherein the formula of the enzyme-labeled diluent is as follows:
Filtering and sterilizing, and preserving at 4 ℃;
3.2 20 x formulation of concentrated washing solution, the formulation of the 20 x concentrated washing solution is: the sterilized 50mL pure water contains NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL;
3.3 Heat shock protein 70 standard gradient solution is prepared as follows:
Preparing a buffer solution containing 5wt% of bovine serum albumin and 0.85wt% of sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate as a standard buffer solution; preparing 7 heat shock protein 70 standard substance gradient solutions with concentration gradients of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL by using a standard substance buffer solution;
3.4 Preparing a sample diluent, wherein the formula of the sample diluent is as follows:
adjusting pH to 7.4, filtering, sterilizing, and preserving at 4deg.C;
3.5 The luminous solution A is carbamide peroxide solution, and the preparation method is as follows:
Dissolving carbamide peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare carbamide peroxide stock solution with the concentration of 0.1mol/L, and gradually diluting to the working concentration of 4.0X10 -3 mol/L by using the disodium hydrogen phosphate-citric acid buffer solution;
3.6 Light-emitting liquid B was a mixture of 3.0X10 -3 mol/L luminol solution and 3.0X10 -3 mol/L p-iodophenol solution at a ratio of 1:1 (v/v).
The preparation method of the luminol solution comprises the following steps: a stock solution of 0.1mol/L luminol was prepared with 0.05mol/L, pH of 10.3 carbonate buffer, left in the dark for three days, and diluted to 3.0X10- -3 mol/L with 0.1mol/L, pH of 10.3 NaHCO 3 -NaOH buffer.
The antibody titer and antibody sensitivity were determined according to the checkerboard method, whereby the points of the standard curve were selected, wherein:
Coating an antibody: the working concentration of the heat shock protein 70 monoclonal antibody 1 (Mab 1) is 10 mug/mL, and the diluent is coating liquid;
The concentrations of each point of the standard curve are 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL respectively, and the diluent is a standard buffer solution.
Example 5
The method for detecting the heat shock protein 70 by using the chemiluminescent immunoassay kit comprises the following steps of:
(1) Sample adding: setting a standard substance hole and a sample hole on a light-emitting plate, adding 50 mu L of sample diluent into the standard substance hole according to a concentration gradient, and then adding 50 mu L of heat shock protein 70 standard substance gradient solution; respectively arranging blank holes and sample holes to be detected in the sample holes, adding 100 mu L of sample diluent into the blank holes, adding 50 mu L of sample diluent into the sample holes to be detected, and then adding 50 mu L of sample to be detected; when in sample adding, a sample is added to the bottom of a hole of the plate, the hole wall is not touched as much as possible, and the mixture is gently shaken and uniformly mixed;
(2) Incubation: incubating for 20 minutes at 37 ℃ after membrane sealing by a sealing plate;
(3) Preparing liquid: diluting 20 times of concentrated washing liquid with distilled water for 20 times to obtain washing liquid for standby;
(4) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, adding 300 mu L of washing liquid into each hole, standing for 30s, discarding, repeating the steps for 5 times, and beating;
(5) Adding enzyme: diluting the Mab2+HRP according to the dilution of 1:50000, wherein the diluent is enzyme-labeled diluent, and 200 mu L of diluted Mab2+HRP solution is added into each hole of the light-emitting plate;
(6) Incubation: incubating for 15 minutes at 37 ℃ after membrane sealing by a sealing plate;
(7) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 20-40 s, discarding, repeating the steps for 5 times, and beating;
(8) And (3) emitting light: adding 50 mu L of luminous liquid A and 50 mu L of luminous liquid B into each hole, gently shaking and uniformly mixing, and developing for 4 minutes at 37 ℃ in a dark place;
(9) And (3) measuring: sequentially measuring the luminous intensity of each hole, namely RLU value, at 425nm wavelength by using a blank Kong Diaoling and a full-automatic chemiluminescence analyzer;
(10) Drawing a standard curve on a coordinate paper by taking each concentration of the standard substance as an abscissa and the RLU value as an ordinate, finding out the corresponding concentration of the sample to be tested according to the measured RLU value by the standard curve, and multiplying the corresponding concentration by a dilution factor to obtain the actual concentration of the sample; or calculating a linear regression equation of the standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample.
And (3) determining the titer and the sensitivity of the enzyme-labeled antibody according to a chessboard method, so as to select points of a standard curve, wherein: enzyme-labeled heat shock protein 70 monoclonal antibody 2: the working titer of the Mab2+HRP is 1:50000, and the diluent is enzyme-labeled diluent.
The detection operation time of the method is only about 40 minutes.
Example 6
The chemiluminescent immunoassay kit of the invention detects linearity, accuracy, sensitivity and precision
1. Linearity of the kit
7 Concentration gradients of heat shock protein 70 standard were used, respectively: heat shock protein 70 standard gradient concentrations of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL, 0ng/mL, RLU value readings (see Table 1) of the standard solutions were performed using the detection method of example 3, and linearity was calculated. The linear assay curves of the kit are shown in FIG. 1.
Table 1: kit linear analysis reading
As can be seen from fig. 1, the curve equation is: y=404.65x+2092.3, and r 2 = 0.9996, it can be seen that the linearity of the kit is good in the range of 0-480 ng/mL.
2. Accuracy of the kit, verification with sample recovery
Shock protein 70 was added to the standard buffer, respectively, such that the final concentration of shock protein 70 was 20ng/mL, 140ng/mL, 420ng/mL, each sample was tested 3 times in parallel, and the calculated recovery statistics are shown in Table 2.
Table 2: recovery rate of heat shock protein 70 standard
The results show that the recovery rate of the heat shock protein 70 standard sample with high, medium and low concentration is between 88.7 and 103.7 percent, the average recovery rate is between 90.5 and 101.1 percent, and the overall average recovery rate is 96.0 percent.
3. Precision of the kit
The heat shock protein 70 standard solution of 120ng/mL was tested in parallel for 10 times, and the statistics of the results are shown in Table 3.
Table 3: precision test results
| Plate hole number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| RLU value | 52436 | 48929 | 53030 | 49750 | 51296 | 50739 | 44896 | 48224 | 50123 | 49013 |
Precision: CV% = 4.66%.
4. Sensitivity of the kit:
Repeating the measurement for 20 times by using a sample diluent (the concentration of the heat shock protein 70 is 0 ng/mL), obtaining a luminescence value corresponding to the average value M and 2 times of the standard deviation SD, substituting the luminescence value into a curve (y=446x+1203) fitted by detection at the first two points (0 and 15 ng/mL) of the standard curve, calculating the detection sensitivity of the method, wherein the calculated concentration is the lowest detection limit of the kit, the lowest detection limit is 0.039ng/mL, and the test is shown in Table 4.
Table 4: sensitivity test results
| Project | Numerical value |
| RLU value: m+2sd (n=20) | 1220 |
| Corresponding concentration value (ng/mL) | 0.039 |
The test result shows that the sensitivity is 0.039ng/mL.
Example 7
The experimental object:
15 patients with hepatocellular carcinoma in the experimental group, 10 men and 5 women, with average age of 40+ -5; healthy control group 15, 8 men, 7 women, average age 42±6.
And (3) measuring: plasma from the experimental group (15 persons) and healthy control group (15 persons) of hepatocellular carcinoma patients was collected, EDTA-Na 2 or heparin was used as an anticoagulant, and centrifugation was performed at 1000 Xg at 4℃for 15 minutes after collection, and immediately after collecting the supernatant, the measurement was performed by the method described in example 5, and the measurement results are shown in Table 5.
Table 5: control and experimental group heat shock protein 70 detection analysis
| Group of | Concentration (ng/mL) | Group of | Concentration (ng/mL) |
| Control group 1 | 19.84 | Experiment group 1 | 41.45 |
| Control group 2 | 20.91 | Experiment group 2 | 35.83 |
| Control group 3 | 19.32 | Experiment group 3 | 38.42 |
| Control group 4 | 19.91 | Experiment group 4 | 30.27 |
| Control group 5 | 20.61 | Experiment group 5 | 45.06 |
| Control group 6 | 20.35 | Experiment group 6 | 34.24 |
| Control group 7 | 19.09 | Experiment group 7 | 41.42 |
| Control group 8 | 19.87 | Experiment group 8 | 33.67 |
| Control group 9 | 20.39 | Experiment group 9 | 36.95 |
| Control group 10 | 19.86 | Experiment group 10 | 32.59 |
| Control group 11 | 19.08 | Experiment group 11 | 40.52 |
| Control group 12 | 19.10 | Experiment group 12 | 33.57 |
| Control group 13 | 20.35 | Experiment group 13 | 35.11 |
| Control group 14 | 19.90 | Experiment group 14 | 38.00 |
| Control group 15 | 20.09 | Experiment group 15 | 31.24 |
As shown in Table 5, the concentration of the heat shock protein 70 in the healthy control group is 19.09-20.91 ng/mL, the concentration of the heat shock protein 70 in the experimental group of the hepatocellular carcinoma patient is 30.27-45.06 ng/mL, which is consistent with the significant increase of the level of the heat shock protein 70 in the blood plasma under the stress state reported in the published literature, thus indicating that the detection kit has the potential application value of clinical detection.
Claims (10)
1. A chemiluminescent immunoassay kit for detecting heat shock protein 70, said kit comprising: the kit comprises a luminescent plate, a heat shock protein 70 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2, an enzyme-labeled diluent, a 20X concentrated washing solution, a luminescent solution A and a luminescent solution B.
2. The chemiluminescent immunoassay kit of claim 1 wherein the luminescent plate is a 96-well or 48-well luminescent plate and each well is coated with heat shock protein 70 monoclonal antibody 1.
3. The chemiluminescent immunoassay kit of claim 1 wherein the heat shock protein 70 standard gradient solution comprises 7 heat shock protein 70 standard solutions having a concentration gradient of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL, 0ng/mL, wherein the heat shock protein 70 standard gradient solution is formulated in a standard buffer that is a 1.0mmol/L disodium hydrogen phosphate-disodium hydrogen phosphate buffer having a ph=7.0 of 5wt% bovine serum albumin, 0.85wt% sodium chloride.
4. The chemiluminescent immunoassay kit of claim 1 wherein the sample diluent is formulated to detect heat shock protein 70 by: comprises NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、SDS 0.1g、 g casein sodium salt, tritonX-100.1 mL, and Proclin300 mL calculated by 1000mL sterilized pure water.
5. The chemiluminescent immunoassay kit of claim 1 wherein the amino acid sequence of heat shock protein 70 monoclonal antibody 1 for an epitope is: DLQQRNSTIQAANLAGLKIL the amino acid sequence of heat shock protein 70 monoclonal antibody 2 against an epitope is: QGGMFLTRAMSGNNKLGGQD; the working titer of the horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2 is 1:10000.
6. The chemiluminescent immunoassay kit of claim 1 wherein the enzyme-labeled diluent is formulated to detect heat shock protein 70 by: the sterilized 1000mL pure water contains 8.0g of NaCl, 2.9g of KH 2PO4 0.2g、Na2HPO4·12H2 O, 93 g of surfactant S, 5g of casein sodium salt, 10g of BSA and 300 mL of Proclin;
the formula of the 20 x concentrated washing liquid is as follows: based on 50mL of sterilized pure water, comprises NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、Tween20 0.5mL.
7. The chemiluminescent immunoassay kit for detecting heat shock protein 70 of claim 1 wherein the luminescent solution A is urea peroxide dissolved in disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.0 to prepare a urea peroxide stock solution of 0.1mol/L, and then the urea peroxide stock solution is gradually diluted to a working concentration of 4.0X10 -3 mol/L with the disodium hydrogen phosphate-citric acid buffer solution; the luminescent solution B is obtained by mixing 3.0X10 -3 mol/L luminol solution with 3.0X10 -3 mol/L p-iodophenol solution according to the ratio of 1:1 (v/v).
8. A method of preparing a chemiluminescent immunoassay kit for detecting heat shock protein 70 of claim 1 comprising the steps of:
1) Preparation of a light-emitting plate:
1.1 Preparing a coating liquid: preparing a phosphate buffer solution with the concentration of 0.01mol/L, and adjusting the pH value to 7.4;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
1.3 Preparing a sealing liquid, wherein the formula and the preparation method of the sealing liquid are as follows:
1.4 Diluting the heat shock protein 70 monoclonal antibody 1 to the working concentration of 10 mug/mL by using a coating liquid, uniformly mixing, and standing for 15 minutes;
1.5 Taking a light-emitting plate, using a 12-channel gun-discharging spot plate to enable the heat shock protein 70 monoclonal antibody to reach 100 mu L/hole, covering a cover plate film, placing in a refrigerator with the temperature of 2-8 ℃ and coating overnight for 20-24 hours;
1.6 Taking out the overnight coated luminescent plate, balancing at room temperature for 30min, throwing away the coating liquid, beating to dry with water-absorbing paper, adding 300 mu L/hole of washing liquid each time, standing at room temperature for 1min, washing for 3 times, and beating to dry with water-absorbing paper;
1.7 200 mu L of sealing liquid is added into each hole, and the mixture is sealed for 16 hours at 2-8 ℃ overnight;
1.8 Taking out the sealed coating plate, balancing at room temperature for 30min, throwing off sealing liquid, and drying with absorbent paper; drying in a silica gel dryer, and maintaining humidity below 30% at room temperature for 5 hr;
1.9 Vacuum packaging with aluminum foil bag, marking, and storing at 2-8deg.C;
2) Preparation of horseradish peroxidase-labeled heat shock protein 70 monoclonal antibody 2
Marking the heat shock protein 70 monoclonal antibody 2 horseradish peroxidase according to a sodium periodate method, and storing the marker at 4 ℃ for later use;
3) Preparing a kit solution:
3.1 Preparing enzyme-labeled diluent, wherein the formula of the enzyme-labeled diluent is as follows:
3.2 20 x formulation of concentrated washing solution, the formulation of the 20 x concentrated washing solution is: the sterilized 50mL pure water contains NaCl 8g、NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL;
3.3 Heat shock protein 70 standard gradient solution is prepared as follows:
Preparing a buffer solution containing 5wt% of bovine serum albumin and 0.85wt% of sodium chloride and having a pH=7.0 and containing 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate as a standard buffer solution; preparing 7 heat shock protein 70 standard substance gradient solutions with concentration gradients of 480ng/mL, 240ng/mL, 120ng/mL, 60ng/mL, 30ng/mL, 15ng/mL and 0ng/mL by using a standard substance buffer solution;
3.4 Preparing a sample diluent, wherein the formula of the sample diluent is as follows: the sterilized 1000mL pure water contains NaCl 8.0g、KH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、SDS 0.1g、.0 g casein sodium salt, triton X-100.1 mL and Proclin300 mL;
3.5 The luminous solution A is carbamide peroxide solution, and the preparation method is as follows:
Dissolving carbamide peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare carbamide peroxide stock solution with the concentration of 0.1mol/L, and gradually diluting to the working concentration of 4.0X10 -3 mol/L by using the disodium hydrogen phosphate-citric acid buffer solution;
3.6 3.0X10 -3 mol/L luminol solution as luminescent solution B and 3.0X10 -3 mol/L p-iodophenol solution are mixed according to the ratio of 1:1 (v/v);
The preparation method of the luminol solution comprises the following steps: a stock solution of 0.1mol/L luminol was prepared with 0.05mol/L, pH of 10.3 carbonate buffer, left in the dark for three days, and diluted to 3.0X10- -3 mol/L with 0.1mol/L, pH of 10.3 NaHCO 3 -NaOH buffer.
9. Use of the chemiluminescent immunoassay kit of claim 1 for detecting heat shock protein 70.
10. The method for detecting heat shock protein 70 by using the chemiluminescent immunoassay kit of claim 1 comprises the following steps:
(1) Sample adding: setting a standard substance hole and a sample hole on a light-emitting plate, adding 50 mu L of sample diluent into the standard substance hole according to a concentration gradient, and then adding 50 mu L of heat shock protein 70 standard substance gradient solution; respectively arranging blank holes and sample holes to be detected in the sample holes, adding 100 mu L of sample diluent into the blank holes, adding 50 mu L of sample diluent into the sample holes to be detected, and then adding 50 mu L of sample to be detected; when in sample adding, a sample is added to the bottom of a hole of the plate, the hole wall is not touched as much as possible, and the mixture is gently shaken and uniformly mixed;
(2) Incubation: incubating for 20 minutes at 37 ℃ after membrane sealing by a sealing plate;
(3) Preparing liquid: diluting 20 times of concentrated washing liquid with distilled water for 20 times to obtain washing liquid for standby;
(4) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, adding 300 mu L of washing liquid into each hole, standing for 30s, discarding, repeating the steps for 5 times, and beating;
(5) Adding enzyme: diluting the Mab2+HRP according to the dilution of 1:50000, wherein the diluent is enzyme-labeled diluent, and 200 mu L of diluted Mab2+HRP solution is added into each hole of the light-emitting plate;
(6) Incubation: incubating for 15 minutes at 37 ℃ after membrane sealing by a sealing plate;
(7) Washing: removing the sealing plate film, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 20-40 s, discarding, repeating the steps for 5 times, and beating;
(8) And (3) emitting light: adding 50 mu L of luminous liquid A and 50 mu L of luminous liquid B into each hole, shaking and mixing uniformly, and developing for 4 minutes at 37 ℃ in a dark place;
(9) And (3) measuring: sequentially measuring the luminous intensity of each hole, namely RLU value, at 425nm wavelength by using a blank Kong Diaoling and a full-automatic chemiluminescence analyzer;
(10) Drawing a standard curve on a coordinate paper by taking each concentration of the standard substance as an abscissa and the RLU value as an ordinate, finding out the corresponding concentration of the sample to be tested according to the measured RLU value by the standard curve, and multiplying the corresponding concentration by a dilution factor to obtain the actual concentration of the sample; or calculating a linear regression equation of the standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample.
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