CN111487413A - Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method - Google Patents

Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method Download PDF

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CN111487413A
CN111487413A CN201910088192.9A CN201910088192A CN111487413A CN 111487413 A CN111487413 A CN 111487413A CN 201910088192 A CN201910088192 A CN 201910088192A CN 111487413 A CN111487413 A CN 111487413A
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detection
fabp
antibody
fatty acid
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王立杰
艾冰花
赵京超
常青侠
陈蒙蒙
武祥伟
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Avioq Biology Technology Co ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01—MEASURING; TESTING
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    • G01N2800/00—Detection or diagnosis of diseases
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    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

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Abstract

The invention belongs to the technical field of in-vitro diagnostic reagents, and provides a detection kit for quantitatively detecting heart-type fatty acid binding protein by an E L ISA method, which comprises an enzyme label plate coated with an anti-H-FABP monoclonal antibody, an anti-H-FABP polyclonal antibody marked by HRP for detection, a standard product diluent, a sample diluent, a concentrated washing solution, a color development solution A, a color development solution B, a stop solution and a detection antibody diluent.

Description

Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to a detection kit for quantitatively detecting heart-type fatty acid binding protein by using an E L ISA method.
Background
Acute Myocardial Infarction (AMI) is a myocardial ischemia necrosis disease caused by acute and persistent ischemia and hypoxia of coronary arteries, is clinically manifested by acute and persistent poststernal pain, can be complicated with arrhythmia, shock or heart failure in severe cases, and has high fatality rate and great harm.
AMI usually has sudden and rapid onset, and early discovery and treatment have great significance for reducing infarct size and protecting cardiac function, so that AMI is a key point for saving the life of a patient and improving the prognosis of the patient.
Heart-type fatty acid binding protein (H-FABP) is a small molecular protein (15.0KD), is a fatty acid carrier in myocardial cells, and is one of the current sensitive markers of early myocardial ischemia injury. Levels in the myocardium were 10 times higher than in skeletal muscle. The levels in the kidney, liver and small intestine are low. Has better specificity to cardiac muscle. After myocardial damage, the sensitivity of myocardial cells to ischemia and hypoxia is increased. The fatty acid needs to be mobilized to supply energy, so that the cardiac fatty acid binding protein level in the myocardial cells is increased, and finally, the level in blood is increased.
When AMI occurs, H-FABP can be detected when myocardial ischemia and hypoxia do not occur, the H-FABP rapidly rises 2H after myocardial damage, reaches a peak value in 6H, and returns to normal within 12-24H, and has higher timeliness compared with other serum biochemical markers of myocardial necrosis such as cTnI, CK-MB and the like.
H-FABP is a protein without enzyme activity, and the qualitative detection and the quantitative detection of the H-FABP both need to be carried out by means of an immunological method. Clinical detection of H-FABP has experienced an increasing detection rate from the earliest radioimmunoassays to the latest latex-microparticle-enhanced immunoturbidimetry and the use of colloidal gold immunoassay in H-FABP assays.
1. Radioimmunoassay
The radioimmunoassay was the first method reported for the quantitative detection of FABP, and in 1974, the radioimmunoassay was used in the study of IFABP, which reacted 14C-labeled antiserum with the specimen to be tested and detected the corresponding I-FABP concentration by measuring the specific activity of radioactivity in the immunoprecipitate, and subsequently, in 1982, the same method was used to complete the quantitative detection of L-FABP.
2. Enzyme immunoassay
(1) Competitive enzyme-linked immunosorbent assay (E L ISA), with the preparation success of H-FABP monoclonal antibody, E L ISA gradually becomes the mainstream method for detecting H-FABP, because H-FABP is relatively small in molecular mass, competitive E L ISA firstly enters into the consideration range of people and is successfully developed by Know lton and the like in 1989, the experiment adopts the mouse anti-rabbit H-FABP monoclonal antibody as a capture antibody, a quantitative peroxidase (HRP) marked standard antigen and the antigen in a specimen are competitively combined with the capture antibody, finally the purpose of antigen quantification in the specimen is achieved through substrate color reaction, the detection time of the method is as long as 16H and cannot meet the requirement of clinical detection, but the success of H-FABP plasma concentration quantification of competitive E L ISA proves the feasibility of E L ISA-based on the monoclonal antibody for H-FABP quantification, and experience is accumulated for the improvement of E L ISA in the future;
(2) the double antibody sandwich E L ISA Ohka ru is equal to the reagent kit for quantitatively detecting H-FABP concentration reported in 1995, which is successfully developed by the double antibody sandwich E L ISA, 8E3 anti-human H-FABP antibody is adopted as a capture antibody, HRP-labeled 8B9 anti-human H-FABP antibody is adopted as a labeled antibody, the test is completed by 4 steps of combining a specimen antigen and the capture antibody, combining an antigen-capture antibody complex and the labeled antibody, adding and developing a substrate and measuring the absorbance at the wavelength of 620nm, the detection time is shortened to 75min, the minimum detection concentration is 1.25 mug/L, the detection linear range of a plasma specimen reaches 220 mug/L1, the precision between the group and the group is respectively lower than 7.9% and 7.0%, the technical parameters show that the reagent kit is satisfactory, then Wodzing is successfully developed by a double one-step method E L ISA for the quantification of the plasma H-FABP concentration, the technology is based on the high affinity of the single-antibody, the single-step method is more clearly developed by the double-step method for detecting the reagent kit for the detection of the plasma H-FABP detection, the detection of the double antibody sandwich ISA L ISA 2, the single-labeled antibody, the detection is very fast and the detection method for the detection of the single-labeled antibody, the detection of the single-labeled antibody, the detection of;
(3) the E L ISA based on in-situ precipitation enhanced elliptic filtration turbidimetry, Robers and 1999 introduce an E L ISA based on in-situ precipitation enhanced elliptic filtration turbidimetry, which can be used for H-FABP quantification, the E L ISA is different from the standard E L ISA in that precipitation substrate Diaminodiphenylammonia (DAB) is used as a substrate of HRP instead of the conventionally used chromogenic substrate Tetramethyldiphenylammonia (TMB), and finally, the turbidity increase condition of the surface of the solid phase carrier is measured by an elliptic filtration enhanced elliptic filtration turbidimetry, and the concentration of H-FABP in the specimen is calculated according to the turbidity increase condition, because the precipitation product of DAB after the action of HRP is mostly concentrated on the surface of the solid phase carrier in situ of the antigen-antibody conjugateTherefore, the turbidity is mainly increased in the two-dimensional structure of the surface of the solid phase carrier, while the color change generated by the classic TMB substrate is distributed in the three-dimensional structure of the reaction hole, and the turbidity is more sensitive than the increase of the turbidity, so that the E L ISA established according to the principle has higher sensitivity and faster reaction time than the prior E L ISA, the minimum detection concentration of H-FABP can reach 100 fmol/L, which is equivalent to 1.25 × 10-4Mu g/L, and the detection time is correspondingly shortened to 10-20 min, but the linear range of the detection concentration is relatively narrow, only 10 mu g/L, and for AMI patient samples with higher concentration, the samples can only enter the linear range of detection through dilution.
3. Application of immunosensor technology
The immunosensor technology combines the specificity of immunological methods with the sensitivity of sensor methods, and can be used for the detection of non-enzymatic proteins.1996, Siegmann-Thoss et al succeeded in designing a 1 st method for detecting the concentration of H-FABP by using an immunosensor, which uses a C lark-type oxygen electrode, uses an anti-H-FABP antibody fixed on a nitrocellulose membrane as a capture antibody, a Glucose Oxidase (GOD) -labeled anti-H-FABP antibody as a labeled antibody, an immune membrane labeled with the labeled antibody is coated on an oxygen electrode, and the concentration of the antigen to be detected is calculated by converting the content of GOD detected by the oxygen electrode after adding the antigen to be detected and a substrate glucose.detection method based on this immunosensor, which requires 30min for detection time, has a detection lower limit of 5 μ g/L, a linear range of 50 μ g/L. however, the immune membrane immobilized with the capture antibody needs to be replaced 10 times with a new one, which is coated on the surface of the electrode, which requires a large amount of manpower and material resources, is not applicable to clinical specimen, and is not applicable to the detection of an antibody, when the detection is carried out by a quantitative detection by a schibenez-based on an enzyme-based on an alkaline phosphatase, which is immobilized antibody, a reference electrode, which is coated with a standard, a substrate, which is not used for detecting an antibody, and is immobilized on a ligand, which is not used for detecting an antibody, and is immobilized in a quantitative detection system, and is immobilized in a quantitative detection method, which is used for detecting an agph sensor, which is not used for detecting an agph, which is not used for detecting an antibody, and is not used for detecting an antibody, and is used for detecting an antibody, which is used for detecting an antibody, and is used for detecting an antibody, which is used for.
4. Latex particle enhanced immunoturbidimetry
The method is characterized in that latex particle enhanced immunoturbidimetry is commonly used for detecting protein with tiny content in 1998, Robers and the like are successfully introduced into H-FABP detection, the latex particles are labeled by 3 anti-H-FABP monoclonal antibodies aiming at different epitopes in experimental design, the antibodies are combined with corresponding antigens to cause the coagulation of the latex particles, and the concentration of the H-FABP is calculated according to the condition of turbidity increase.
5. Application of immune colloidal gold technology
The test paper strip is composed of 3 areas, namely (1) a sample receiving area consisting of a solid-phase plasma separation medium, (2) a reaction area consisting of a combination plate containing a chromogenic antibody marked by colloidal gold, and (3) a detection area consisting of a nylon membrane marked with 2 capture antibodies and a cellulose membrane with strong water absorption capacity, wherein the test paper strip is characterized in that the solid-phase plasma separation medium is designed to separate plasma in an anticoagulated whole blood specimen, the design clinically omits the step of sample centrifugation, indirectly shortens the detection time, and the test shows that the qualitative time of the whole blood specimen based on the immune colloidal gold technology is within 15min, the operation is convenient, the test paper strip is only incapable of quantitative determination, limits the application of the H-FABP in the aspects of myocardial area estimation and dynamic observation, and only needs to be calculated by a special test method of a special test paper strip with a constant density of Chan, and a constant density of an immune colloidal gold-labeled with specific monoclonal antibody, namely a constant density, equal to the concentration of the H-FABP within 15min, and a constant density range of a constant density, namely equal to the measured sample within 15 g-868, and equal to the standard of a constant density, namely equal to the standard of a constant density, namely equal to detect the H-10 linear infarction, and equal to detect the sample within 15 g of a constant test paper strip, wherein the test paper strip is designed to detect the sample within 15.
AM1 is a clinical emergency seriously endangering the life of a patient, rapid diagnosis and timely decision are important guarantees for saving the life of the patient, detection at rescue places or detection beside beds have important values in the diagnosis of the diseases, the invention aims to construct an in vitro diagnosis kit for quantitatively detecting the concentration level of the heart-type fatty acid binding protein in human serum by adopting a double-antibody sandwich E L ISA method, and the in vitro diagnosis kit can conveniently and rapidly carry out exclusion screening on AMI patients in the early stage of morbidity.
Disclosure of Invention
The embodiment of the invention provides a detection kit for quantitatively detecting heart-type fatty acid binding protein by an E L ISA method, and aims to solve the problems of poor uniformity, low stability, low specificity and long detection time of the existing kit.
The embodiment of the invention is realized in such a way that a detection kit for quantitatively detecting heart-type fatty acid binding protein by an E L ISA method comprises an ELISA plate coated with an anti-H-FABP monoclonal antibody, an HRP-labeled anti-H-FABP polyclonal antibody for detection, a standard substance diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.
The preparation method of the ELISA plate coated with the anti-H-FABP monoclonal antibody comprises the following steps:
1) dissolving the coated anti-H-FABP monoclonal antibody in a coating buffer solution until the concentration of the coated antibody is 2ug/m L to obtain a mixed solution;
2) adding the mixed solution obtained in the step 1) into the holes of a microporous plate, wherein each hole is 0.1m L, covering a plate membrane, placing for 18-24 hours at the temperature of 2-8 ℃, washing for 5 times by using 0.5% Tween20-PBS, adding a sealing solution into each hole, wherein each hole is 0.12m L, placing for 18-24 hours at the temperature of 2-8 ℃, washing for 5 times by using 0.5% Tween20-PBS, placing in a drying room at the temperature of 25-35 ℃, the humidity is less than 40%, the drying time is 20-24 hours, packaging plates by using aluminum foil bags, and storing at the temperature of 2-8 ℃ to obtain the enzyme-labeled plate.
The coated anti-H-FABP monoclonal antibody is an anti-H-FABP monoclonal antibody and is purchased from HyTest company of Finland.
Composition of the coating buffer: anhydrous sodium carbonate 1.59 g; NaHCO 232.98g, 0.01g of amaranth and purified water with the volume of 1000m L;
the formula of the sealing liquid is as follows: na (Na)2HPO4·12H2O 5.20g；NaH2PO4·2H20.87g of O, 20.00g of cane sugar, 10.00g of casein, 10.00g of BSA and purified water with the volume of 1000m L.
Among the anti-H-FABP polyclonal antibodies for HRP labeling detection, the anti-H-FABP polyclonal antibody is purchased from HyTest company of Finland.
The preparation method of the anti-H-FABP polyclonal antibody for detecting the HRP mark comprises the following steps:
(1) dialyzing the anti-H-FABP polyclonal antibody in 0.01M carbonate buffer solution with pH of 9.0-9.5 to obtain final concentration of 5mg/M L;
(2) 10mg of HRP was dissolved in 2m L H2O, the final concentration is 5mg/m L, and an HRP solution is obtained;
(3) adding NaIO with the concentration of 37.5mg/m L and 0.4m L into the HRP solution obtained in the step (2)4Stirring and oxidizing the solution at room temperature in a dark place for 20 min;
(4) dialyzing the oxidized enzyme solution at 4 ℃ in 1mM acetate buffer solution with pH4.4 overnight, dialyzing with cut-off molecular weight of 12000-14000, and changing the solution for 3 times;
(5) taking out the solution in the dialysis bag, and adding 0.2M Na2CO3Adjusting the pH of the buffer solution to 9.0-9.5, quickly mixing the buffer solution with the antibody 1:1 with the pH of 9.0 obtained in the step (1), and slightly stirring the mixture at room temperature in a dark place for 2 hours;
(6) adding new 6mg/m L NaBH4Mixing 100ul of solution, and reducing for 2h at 4 ℃;
(7) dialyzing the reduced reaction system in 0.15M PBS (pH7.4PBS) at 4 ℃ overnight, and changing the solution for 3 times;
(8) adding BSA to make the final concentration 2-3mg/m L, mixing, 10000rpm × 3min, collecting supernatant, packaging, and freezing to obtain the anti-H-FABP polyclonal antibody for detecting HRP mark.
The standard substance is H-FABP antigen and is purchased from HyTest company of Finland;
the standard substance diluent comprises the following components: the solvent was 20mM, pH7.4PBS buffer, and the solutes and their concentrations in the standard dilutions were as follows: 1 percent (mass percentage content) casein, 8 percent (mass percentage content) trehalose, 3 percent (mass percentage content) mannitol, 1mM EDTA, 0.5 percent (mass percentage content) glycine, 150mM NaCl and 0.1 percent (mass percentage content) preservative Proclin-300.
Composition of the sample dilution: the solvent was 20mM PBS buffer pH6.0, and the solutes and their mass concentrations in the sample dilution were as follows: 0.2% of gelatin, 0.2% of casein, 150mM of NaCl, 0.01% of Tween-20, 5% of sucrose, 0.5/ten thousand of bromopotash phenol purple and 0.1% of preservative Proclin-300.
The concentrated washing solution comprises water as solvent, and 116 g/L Na as solute and its concentration in the concentrated washing solution2HPO412H2O，11.84g/L NaH2PO42H2O, 180 g/L NaCl, 5m L/L Tween20, 1m L/L preservative Proclin-300.
The color developing solution A comprises a solvent which is water, and solutes and the concentrations of the solutes in the color developing solution A are 7.28 g/L citric acid, 23.75 g/L disodium hydrogen phosphate and 0.5 g/L urine of hydrogen peroxide.
The color developing solution B comprises water as solvent, 100m L/L methanol as solute, 1 g/L PVA and 1.5 g/L Na2S2O31m L/L HCl, 0.03% (mass percent) TMB and 1% (mass percent) DMSO.
The stop solution comprises water as solvent, 200m L/1.8L sulfuric acid and 1.35 g/1.8L EDTA as solute and the concentration of the solute in the stop solution is shown in the specification.
The composition of the detection antibody diluent is as follows: the solvent was 20mM, pH7.4PBS buffer, and the solutes and their concentrations in the assay antibody dilution were as follows: 2% (mass percent) BSA, 0.1% (mass percent) casein, 150mM NaCl, 0.01% (mass percent) Tween20, 5/1 ten thousand (mass percent) amino biquinol, 1/20 thousand (mass percent) dye food red, and 0.1% (mass percent) preservative Proclin-300.
The beneficial effects achieved by the invention are mainly embodied in the following aspects:
1. the detection kit for quantitatively detecting the heart-type fatty acid binding protein by the E L ISA method has high sensitivity, the minimum detection limit of the kit is as low as 1pg/m L, the kit has good uniformity and high stability, the intra-batch variation coefficient is less than 10 percent, and the inter-batch variation coefficient is less than 5 percent.
2. The kit has good specificity, has very low cross reaction with serum total bilirubin TBI L and triglyceride TG which are easy to interfere with the test in serum, and cannot interfere with the measurement of H-FABP.
3. The kit can realize rapid quantitative detection of H-FABP in clinical serum samples so as to be used for simple, convenient and rapid exclusivity screening of AMI patients in early disease.
Drawings
FIG. 1 is a standard graph obtained by the test in the second embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example composition of a kit:
the embodiment of the invention provides a detection kit for quantitatively detecting heart-type fatty acid binding protein by an E L ISA method, which comprises an ELISA plate coated with an anti-H-FABP monoclonal antibody, an HRP-labeled anti-H-FABP polyclonal antibody for detection, a standard substance diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.
The components of the materials contained in the kit and preparation were as follows:
the preparation method of the ELISA plate coated with the anti-H-FABP monoclonal antibody comprises the following steps:
1) dissolving the coated anti-H-FABP monoclonal antibody in a coating buffer solution until the concentration of the coated antibody is 2ug/m L to obtain a mixed solution;
2) adding the mixed solution obtained in the step 1) into the holes of a microporous plate, wherein each hole is 0.1m L, covering a plate membrane, placing for 18-24 hours at the temperature of 2-8 ℃, washing for 5 times by using 0.5% Tween20-PBS, adding a sealing solution into each hole, wherein each hole is 0.12m L, placing for 18-24 hours at the temperature of 2-8 ℃, washing for 5 times by using 0.5% Tween20-PBS, placing in a drying room at the temperature of 25-35 ℃, the humidity is less than 40%, the drying time is 20-24 hours, packaging plates by using aluminum foil bags, and storing at the temperature of 2-8 ℃ to obtain the enzyme-labeled plate.
The coated anti-H-FABP monoclonal antibody is an anti-H-FABP monoclonal antibody and is purchased from HyTest company of Finland.
Composition of the coating buffer: anhydrous sodium carbonate 1.59 g; NaHCO 232.98g, 0.01g of amaranth and purified water with the volume of 1000m L;
the formula of the sealing liquid is as follows: na (Na)2HPO4·12H2O 5.20g；NaH2PO4·2H20.87g of O, 20.00g of cane sugar, 10.00g of casein, 10.00g of BSA and purified water with the volume of 1000m L.
Among the anti-H-FABP polyclonal antibodies for HRP labeling detection, the anti-H-FABP polyclonal antibody is purchased from HyTest company of Finland.
The preparation method of the anti-H-FABP polyclonal antibody for detecting the HRP mark comprises the following steps:
(1) dialyzing the anti-H-FABP polyclonal antibody in 0.01M carbonate buffer solution with pH of 9.0-9.5 to obtain final concentration of 5mg/M L;
(2) 10mg of HRP was dissolved in 2m L H2O, the final concentration is 5mg/m L, and an HRP solution is obtained;
(3) adding NaIO with the concentration of 37.5mg/m L and 0.4m L into the HRP solution obtained in the step (2)4Stirring and oxidizing the solution at room temperature in a dark place for 20 min;
(4) dialyzing the oxidized enzyme solution at 4 ℃ in 1mM acetate buffer solution with pH4.4 overnight, dialyzing with cut-off molecular weight of 12000-14000, and changing the solution for 3 times;
(5) taking out the solution in the dialysis bag, and adding 0.2M Na2CO3Adjusting the pH of the buffer solution to 9.0-9.5, quickly mixing the buffer solution with the antibody 1:1 with the pH of 9.0 obtained in the step (1), and slightly stirring the mixture at room temperature in a dark place for 2 hours;
(6) adding new 6mg/m L NaBH4Mixing 100ul of solution, and reducing for 2h at 4 ℃;
(7) dialyzing the reduced reaction system in 0.15M PBS (pH7.4PBS) at 4 ℃ overnight, and changing the solution for 3 times;
(8) adding BSA to make the final concentration 2-3mg/m L, mixing, 10000rpm × 3min, collecting supernatant, packaging, and freezing to obtain the anti-H-FABP polyclonal antibody for detecting HRP mark.
The standard is H-FABP antigen and is purchased from HyTest company of Finland.
Composition of the sample dilution: the solvent was 20mM PBS buffer pH6.0, and the solutes and their mass concentrations in the sample dilution were as follows: 0.2% of gelatin, 0.2% of casein, 150mM of NaCl, 0.01% of Tween-20, 5% of sucrose, 0.5/ten thousand of bromopotash phenol purple and 0.1% of preservative Proclin-300.
The concentrated washing solution comprises water as solvent, and 116 g/L Na as solute and its concentration in the concentrated washing solution2HPO412H2O，11.84g/L NaH2PO4·2H2O, 180 g/L NaCl, 5m L/L Tween20, 1m L/L preservative Proclin-300.
The color developing solution A comprises a solvent which is water, and solutes and the concentrations of the solutes in the color developing solution A are 7.28 g/L citric acid, 23.75 g/L disodium hydrogen phosphate and 0.5 g/L urine of hydrogen peroxide.
The color developing solution B comprises water as solvent, 100m L/L methanol as solute, 1 g/L PVA and 1.5 g/L Na2S2O31m L/L HCl, 0.03% (mass percent) TMB and 1% (mass percent) DMSO.
The stop solution comprises water as solvent, 200m L/1.8L sulfuric acid and 1.35 g/1.8L EDTA as solute and the concentration of the solute in the stop solution is shown in the specification.
The standard substance diluent comprises the following components: the solvent was 20mM, pH7.4PBS buffer, and the solutes and their concentrations in the standard dilutions were as follows: 1 percent (mass percentage content) casein, 8 percent (mass percentage content) trehalose, 3 percent (mass percentage content) mannitol, 1mM EDTA, 0.5 percent (mass percentage content) glycine, 150mM NaCl and 0.1 percent (mass percentage content) preservative Proclin-300.
The composition of the detection antibody diluent is as follows: the solvent was 20mM, pH7.4PBS buffer, and the solutes and their concentrations in the assay antibody dilution were as follows: 2% (mass percent) BSA, 0.1% (mass percent) casein, 150mM NaCl, 0.01% (mass percent) Tween20, 5/1 ten thousand (mass percent) amino biquinol, 1/20 thousand (mass percent) dye food red, and 0.1% (mass percent) preservative Proclin-300.
Example two: step of detection with the kit
(I): preparation of Standard Curve
The following solutions were prepared before use:
(1) diluting an HRP-labeled detection anti-H-FABP polyclonal antibody with a detection antibody diluent so that the ratio of a solute (the detection anti-H-FABP polyclonal antibody) to a solvent (the detection antibody diluent) is 1: 5000;
(2) and dissolving the standard substance by using the standard substance diluent to ensure that the concentration of the standard substance is 0pg/m L, 5pg/m L, 15pg/m L, 40pg/m L, 130pg/m L and 300pg/m L in sequence.
The detection steps are as follows:
1. preparing a solution, namely diluting a concentrated washing solution (20 ×) with distilled water or deionized water by 1:20 for later use (30m L washing solution is diluted to 600m L, and 50m L washing solution is diluted to 1000m L);
2. numbering: numbering the micropores corresponding to the sample in sequence;
3. loading, namely respectively adding 50 mu L of calibrator solution into corresponding holes, and lightly shaking and uniformly mixing;
4. and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 30 minutes at 37 ℃;
5. washing: drying the liquid in the holes, fully washing for 5 times by using a washing liquid, and drying;
6. adding enzyme, namely adding 100 mu L of enzyme conjugate into each hole, and lightly shaking and uniformly mixing;
7. and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 30 minutes at 37 ℃;
8. washing: fully washing for 5 times by using a washing solution, and drying;
9. developing, namely adding 50 mu L of developing solution A, B into each hole, gently shaking and uniformly mixing, and developing for 15 minutes at 37 ℃ in a dark place;
10. adding 50 mu L of stop solution into each hole, tapping and mixing uniformly, setting the wavelength of an enzyme-labeling instrument at 450nm (detection with dual wavelength of 450/630nm is recommended), adjusting the zero point by using a blank hole, and then measuring the OD value of each hole;
11. data processing:
data processing was performed using a log-log linear fit. The logarithm of the OD value of each calibrator (the OD value of S0 was subtracted) was plotted on the ordinate (Y-axis) and the logarithm of the concentration of each calibrator was plotted on the abscissa (X-axis) to obtain a calibration curve. The resulting standard curve is shown in FIG. 1.
The equation for the standard curve is that y is 0.6572x-1.1613 with a correlation coefficient of 0.9994.
(II) detection of sample to be detected
In step 3, a sample solution to be detected is added, and in step 11, the OD value of the sample solution to be detected is substituted into the standard curve to obtain the content of H-FABP in the sample solution to be detected. The remaining steps were the same as in experiment one.
The sample to be detected is fresh serum or fresh plasma sample, and is separated within 24 hours after the venous blood collection. Serum plasma samples should not be stored at 4 ℃ for more than 1 week. If the measurement can not be carried out within 1 week after blood sampling, the serum sample is sealed and placed below-20 ℃ to avoid repeated freeze thawing. Severely hemolyzed, lipemic samples were not available for detection.
EXAMPLE III kit Properties
The kit to be detected is as follows: three batches of kit (batch one, batch two and batch three)
(one) minimum detection limit
1. And adding 50 mu L standard substance into each well of a microplate coated with H-FABP, gently shaking and uniformly mixing, incubating at 37 ℃ for 30 minutes, washing the plate for 5 times, adding 100 mu L enzyme conjugate into each well, incubating at 37 ℃ for 30 minutes, arranging 20 multiple wells for each kit to be tested and S0 solution, and adding double wells for other calibrator.
2. After completing step 1, fully washing with washing solution for 5 times, and drying.
3. After the step 2 is completed, 50 mu L color development liquid A and 50 mu L color development liquid B are added into each hole, light-shielding color development is carried out for 15 minutes at 37 ℃, stop solution is added into each hole for 50 mu L, detection is carried out on an enzyme-linked immunosorbent assay by using 450/630nm dual-wavelength, and the light absorption value of each hole is shown in table 1.
Calculating the Mean (Mean) and Standard Deviation (SD) of the absorbance values corresponding to the 50 mu L S0 solution of 20 wells, and calculating the concentration value of Mean +2 × SD as the lowest detection limit by a fitting equation.
Table 1: three-batch detection of light absorption value of each well
Figure BDA0001962450880000131
Figure BDA0001962450880000141
From the data in Table 1, it is clear that the minimum detection limit of the three batches of kits is below 1pg/m L.
(II) repeatability and run-to-run differences
The repeatability is the Coefficient of Variation (CV) obtained by measuring a sample by using the same batch of kit, each quality control product needs to be subjected to 10-hole precision measurement randomly in one plate, the average concentration (Mean) and the Standard Deviation (SD) of the measurement result are calculated, and the Coefficient of Variation (CV) in the batch is SD/Mean × 100%
The inter-batch difference is the repeatability among different batches of kits, three batches of kits are randomly drawn, the quality control substances are measured by the kits for 3 times, the average concentration (Mean) and the Standard Deviation (SD) of the measurement results are calculated, and the inter-batch variation Coefficient (CV) is 100 percent of SD/Mean × 100 percent.
The quality control product is H-FABP.
1. Adding 50 mu L specific solution into each well of a microplate coated with H-FABP, gently shaking and uniformly mixing, incubating at 37 ℃ for 30 minutes, washing the plate for 5 times, adding 100 mu L into each well, and incubating at 37 ℃ for 30 minutes.
The specific solution is a specific solution A or a specific solution B.
The specific solution A is S0 solution, S1 solution, S2 solution, S3 solution, S4 solution or S5 solution. Two duplicate wells were provided for each specific solution A in each test kit.
The specific solution B is a quality control solution with different concentrations.
The preparation method of the quality control solution comprises the steps of dissolving a quality control in 20mM PBS (phosphate buffer solution) with the pH value of 7.4 to obtain a low-concentration quality control solution (QC1) of 100pg/m L and a high-concentration quality control solution (QC2) of 500pg/m L, and each specific solution B is provided with 10 multiple holes.
2. After completing step 1, fully washing with washing solution for 5 times, and drying.
3. After the step 2 is completed, 50 mu L color development liquid A and 50 mu L color development liquid B are added into each hole, light-shielding color development is carried out for 15 minutes at 37 ℃, stop solution is added into each hole for 50 mu L, and detection is carried out on a microplate reader by using 450/630nm double-wavelength.
The absorbance of each well when the particular solution A was used is shown in Table 2.
TABLE 2 Absorbance values for wells measured with specific solution A
Figure BDA0001962450880000142
Figure BDA0001962450880000151
The absorbance of each well when the specified solution B was used is shown in Table 3.
TABLE 3 Absorbance values for wells measured with specific solution B
QC1 QC2 QC1 QC2 QC1 QC2
1 0.718 1.818 0.774 1.900 0.773 1.805
2 0.745 1.956 0.705 1.827 0.763 1.851
3 0.785 1.843 0.775 1.875 0.751 1.901
4 0.706 1.882 0.468 1.714 0.764 1.953
5 0.762 1.940 0.466 1.720 0.708 1.840
6 0.742 1.930 0.404 1.756 0.712 1.881
7 0.755 1.840 0.740 1.707 0.718 1.837
8 0.703 1.877 0.719 1.679 0.719 1.872
9 0.716 1.920 0.747 1.611 0.747 1.860
10 0.753 1.802 0.715 1.910 0.764 1.953
Data processing was performed using a log-log linear fit. The H-FABP concentration in the specified solution B was calculated from the standard curve. The results are shown in tables 4 and 5. three batches of the kit determine both high and low concentrations of quality control. The intra-batch variation coefficients are all less than 10%, which shows that the uniformity of the kit is good, and the result has repeatability.
TABLE 4
QC1 QC2 QC1 QC2 QC1 QC2
1 104.78 561.35 112.32 557.05 112.36 518.78
2 101.23 535.41 95.06 519.50 109.76 591.03
3 97.50 556.65 112.57 544.06 105.14 531.57
4 97.25 557.17 106.42 582.90 115.51 543.97
5 100.48 541.07 109.74 526.11 109.50 572.32
6 97.99 567.13 95.78 545.10 104.38 544.48
7 100.48 594.79 96.75 508.41 103.88 528.49
8 114.12 556.12 98.22 578.11 95.45 546.56
9 105.29 577.17 98.46 572.81 105.65 540.34
10 109.92 516.05 97.48 562.28 110.02 589.42
Mean value of 102.90 556.29 102.28 549.63 107.17 550.70
SD 5.62 21.98 7.14 25.56 5.57 25.12
CV 5.5％ 4.0％ 7.0％ 4.7％ 5.2％ 4.6％
TABLE 5
QC1 QC2
First batch 102.90 556.29
Second batch 102.28 549.63
Third batch 107.17 550.70
Mean value of 104.12 552.21
SD 2.17 2.92
CV 2.1％ 0.5％
Three batches of kits are used for measuring two quality control products with high concentration and low concentration, and the batch-to-batch variation coefficient is less than 5 percent, which shows that the kits in different batches have small variation and the measurement result has repeatability.
In summary, the main performance indexes of the kit provided by the invention have the following standards:
the lowest detection limit is not higher than 1pg/m L;
repeatability: the intra-batch variation coefficient is not higher than 10%;
inter-batch difference: the inter-batch coefficient of variation is not higher than 5%.
(III) specificity
1. Adding 50 mu L specific solution into each well of a microplate coated with H-FABP, gently shaking and uniformly mixing, incubating at 37 ℃ for 30 minutes, washing the plate for 5 times, adding 100 mu L into each well, and incubating at 37 ℃ for 30 minutes.
The specific solutions were S0 solution, S1 solution, S2 solution, S3 solution, S4 solution, S5 solution, sample solution with serum total bilirubin TBI L of 40. mu. mol/L and sample solution with triglyceride TG of 4 mmol/L.
2. After completing step 1, fully washing with washing solution for 5 times, and drying.
3. After the step 2 is completed, 50 mu L color development liquid A and 50 mu L color development liquid B are added into each hole, light-shielding color development is carried out for 15 minutes at 37 ℃, stop solution is added into each hole for 50 mu L, and detection is carried out on a microplate reader by using 450/630nm double-wavelength.
L og-L og fit was made based on the OD values of the calibration points and the corresponding concentrations, the measured H-FABP values of the cross-reactive substances were calculated from the standard curve, which are the crossing values of the reagent with TBI L and the crossing values of the reagent with TG.
The absorbance and back-extrapolated concentration for each well are shown in Table 6.
TABLE 6 analysis of the results of the specificity experiment
Figure BDA0001962450880000171
Figure BDA0001962450880000181
The substances which easily interfere with the measurement of H-FABP in human blood are serum total bilirubin TBI L and triglyceride TG., which were tested with high concentrations of these substances, and the results shown in Table 7 indicate that the kit has low cross-reactivity with these substances and does not interfere with the measurement of H-FABP.
TABLE 7
Reactive substance Concentration of Cross reaction value
Serum total bilirubin (TBI L) 40μmol/mL <5pg/mL
Triglycerides (TG) 4mmol/L <5pg/mL
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A detection kit for quantitatively detecting heart-type fatty acid binding protein by an E L ISA method is characterized by comprising an ELISA plate coated with an anti-H-FABP monoclonal antibody, an HRP-labeled anti-H-FABP polyclonal antibody for detection, a standard diluent, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B, a stop solution and a detection antibody diluent.
2. The detection kit for quantitatively detecting the cardioid fatty acid binding protein by the E L ISA method according to claim 1, wherein the preparation method of the ELISA plate coated with the anti-H-FABP monoclonal antibody comprises the following steps:
1) dissolving the coated anti-H-FABP monoclonal antibody in a coating buffer solution until the concentration of the coated antibody is 2ug/m L to obtain a mixed solution;
2) adding the mixed solution obtained in the step 1) into the holes of a microporous plate, wherein each hole is 0.1m L, covering a plate membrane, placing for 18-24 hours at the temperature of 2-8 ℃, washing for 5 times by using 0.5% Tween20-PBS, adding a sealing solution into each hole, wherein each hole is 0.12m L, placing for 18-24 hours at the temperature of 2-8 ℃, washing for 5 times by using 0.5% Tween20-PBS, placing in a drying room at the temperature of 25-35 ℃, the humidity is less than 40%, the drying time is 20-24 hours, packaging plates by using aluminum foil bags, and storing at the temperature of 2-8 ℃ to obtain the enzyme-labeled plate.
3. The detection kit for quantitatively detecting the cardiac fatty acid binding protein by the E L ISA method of claim 2, wherein the coating buffer comprises 1.59g of anhydrous sodium carbonate and NaHCO32.98g, 0.01g of amaranth and purified water with the volume of 1000m L;
the formula of the sealing liquid is as follows: na (Na)2HPO4·12H2O 5.20g；NaH2PO4·2H20.87g of O, 20.00g of cane sugar, 10.00g of casein, 10.00g of BSA and purified water with the volume of 1000m L.
4. The detection kit for quantitatively detecting the cardioid fatty acid binding protein according to the E L ISA method of claim 1, wherein the preparation method of the anti-H-FABP polyclonal antibody for HRP labeling detection comprises the following steps:
(1) dialyzing the anti-H-FABP polyclonal antibody in 0.01M carbonate buffer solution with pH of 9.0-9.5 to obtain final concentration of 5mg/M L;
(2) dissolving 10mg of HRP in 2m L H2O to obtain an HRP solution with the final concentration of 5mg/m L;
(3) adding 0.4m of NaIO4 solution with L concentration of 37.5mg/m L into the HRP solution obtained in the step (2), and stirring and oxidizing for 20min at room temperature in the dark;
(4) dialyzing the oxidized enzyme solution at 4 ℃ in 1mM acetate buffer solution with pH4.4 overnight, dialyzing with cut-off molecular weight of 12000-14000, and changing the solution for 3 times;
(5) taking out the solution in the dialysis bag, adding 0.2M Na2CO3 buffer solution, adjusting pH to 9.0-9.5, rapidly mixing with the antibody 1:1 with pH9.0 obtained in step (1), and slightly stirring at room temperature in the dark for 2 h;
(6) adding 100ul of newly prepared 6mg/m L NaBH4 solution, uniformly mixing, and carrying out reaction reduction for 2h at 4 ℃;
(7) dialyzing the reduced reaction system in 0.15M PBS (pH7.4PBS) at 4 ℃ overnight, and changing the solution for 3 times;
(8) adding BSA to make the final concentration 2-3mg/m L, mixing, 10000rpm × 3min, collecting supernatant, packaging, and freezing to obtain the anti-H-FABP polyclonal antibody for detecting HRP mark.
5. The test kit for quantitatively detecting the cardiac fatty acid binding protein according to the E L ISA method of claim 1, wherein the standard substance is H-FABP antigen purchased from HyTest corporation of Finland;
the standard substance diluent comprises the following components: the solvent was 20mM, pH7.4PBS buffer, and the solutes and their concentrations in the standard dilutions were as follows: casein accounting for 1 percent by mass, trehalose accounting for 8 percent by mass, mannitol accounting for 3 percent by mass, 1mM EDTA, glycine accounting for 0.5 percent by mass, 150mM NaCl and a preservative Proclin-300 accounting for 0.1 percent by mass.
6. The kit for the quantitative determination of heart-type fatty acid binding protein according to the E L ISA method of claim 1, wherein the sample diluent comprises 20mM PBS buffer solution with pH6.0 as solvent, 0.2% gelatin, 0.2% casein, 150mM NaCl, 0.01% Tween-20, 5% sucrose, 0.5/ten thousand potassium bromate phenol violet, and 0.1% preservative Proclin-300 as solute and the mass concentration thereof in the sample diluent.
7. The kit for quantitatively detecting heart-type fatty acid binding protein according to the E L ISA method of claim 1, wherein the concentrated washing solution comprises water as a solvent, and 116 g/L Na2HPO 412H 2O, 11.84 g/L NaH2PO 42H 2O, 180 g/L NaCl, 5m L/L Tween20, and 1m L/L preservative Proclin-300 as solutes.
8. The kit for quantitatively detecting heart-type fatty acid binding protein according to the E L ISA method of claim 1, wherein the color-developing solution A consists of water as a solvent, 7.28 g/L citric acid as a solute, 23.75 g/L disodium hydrogen phosphate as a disodium hydrogen phosphate, and 0.5 g/L hydrogen peroxide as a urine solution;
the color developing solution B comprises a solvent which is water, solutes and the concentrations of the solutes in the color developing solution B are 100m L/L methanol, 1 g/L PVA, 1.5 g/L Na2S2O3, 1m L/L HCl, 0.03 percent of TMB and 1 percent of DMSO.
9. The kit for detecting the amount of cardiac fatty acid binding protein according to the ISA method of claim 1, wherein the stop buffer comprises water as a solvent, 200m L/1.8L sulfuric acid as a solute, and 1.35 g/1.8L EDTA as a concentration in the stop buffer.
10. The kit for quantitatively detecting heart-type fatty acid binding protein according to the E L ISA method of claim 1, wherein the diluent for the detection antibody comprises 20mM PBS buffer solution with pH7.4 as a solvent, 2% BSA as a solute and the concentration thereof in the diluent for the detection antibody, 0.1% casein as a mass percentage, 150mM NaCl as a mass percentage, 0.01% Tween20 as a mass percentage, 5/1% aminopyrine as a mass percentage, 1/20% dye food red as a mass percentage, and 0.1% Proclin-300 as a mass percentage.
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