CN110716052A - Human eosinophil cationic protein and myeloperoxidase detection kit and application thereof - Google Patents
Human eosinophil cationic protein and myeloperoxidase detection kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a human eosinophil cationic protein and myeloperoxidase detection kit and application thereof. The two kits of the invention adopt a reaction mode of a double-antibody sandwich one-step method, and can simultaneously carry out very specific quantitative and qualitative detection on human eosinophilic granulocyte cationic protein and myeloperoxidase molecules in patient serum and nasal secretion samples. The method not only effectively utilizes the technical principle of chemiluminescence, but also applies an enzyme-catalyzed luminescent substrate on the basis of enzyme-linked immunoassay, improves the detection sensitivity, has the advantages of high stability, good specificity, good accuracy, simple operation and no radioactive pollution, and can provide a more specific, rapid and reliable basis for clinical diagnosis of inflammatory diseases such as bronchial asthma, nasosinusitis, rhinitis and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a human eosinophil cationic protein and myeloperoxidase detection kit and application thereof.
Background
Allergic Rhinitis (AR) and Chronic Rhinosinusitis (CRS) are the most common diseases in ear, nose, throat, head and neck surgery. Often the diagnosis depends on the symptoms and signs of the patient. The cytological examination of nasal secretion is used as an important auxiliary diagnosis method, is beneficial to the accurate diagnosis and individualized treatment of rhinitis patients, and has not wide application although being developed clinically because of complicated operation and lack of uniform standard. In our previous nasal secretion cytology studies, it can be determined that eosinophils and neutrophils account for the absolute majority (> 98%) of nasal secretions, and that nasal secretions of patients with AR are mainly characterized by eosinophils, and nasal secretions of patients with CRS are mainly characterized by neutrophils. The accuracy of the results is affected because the sampling process of the nasal cytology examination is unstable and can be affected and restricted by the condition of the patient at the time of the visit (such as the secretion amount is too small, the patient is not matched, etc.). In contrast, nasal secretions are more easily and stably obtained. Eosinophil Cationic Protein (ECP) is a basic protein, is an important index reflecting the activation amount of eosinophils, and is one of the markers of clinical examination of allergic patients. Myeloperoxidase (MPO) is a heme protease containing a heme prosthetic group and is also the most abundant pro-inflammatory enzyme stored in neutrophils, reflecting the activation of neutrophils.
At present, no product for judging the nature of nasal inflammation exists in clinic, no competitive product exists, and even no product exists. The judgment of the nasal cavity inflammation and the adjustment of the treatment are carried out by doctors completely depending on experience. ECP and MPO have wide and important application value clinically; the nasal cavity local inflammation is accurately judged, so that the rhinitis is accurately treated, and the treatment effect is objectively evaluated.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a human eosinophil cationic protein and myeloperoxidase detection kit and application thereof.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a kit for detecting cationic protein and myeloperoxidase of human eosinophils comprises the following two types:
A) chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) of human eosinophil cationic protein and myeloperoxidase, which comprises the following components:
① human eosinophil cationic protein and human myeloperoxidase standard;
② a vector coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody;
③ labeled human eosinophil cationic protein monoclonal antibody and human myeloperoxidase monoclonal antibody, ③ labeled antibody and ② coating antibody are paired antibodies which respectively form a double-anti-sandwich structure with human eosinophil cationic protein and human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein paired antibody and the human myeloperoxidase paired antibody;
④ substrate for color development;
B) an immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, comprising:
① sample loading pad adhered to one end of the bottom plate;
② a colloidal gold pad containing a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody, which is tightly pressed against the loading pad;
③ nitrocellulose NC membrane tightly pressed with one end of the colloidal gold pad, the nitrocellulose NC membrane is coated with a detection line T and a quality control line C which are separated from each other, the detection line T is coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody which are matched with the ② labeled antibody, the quality control line C is coated with a goat anti-mouse IgG antibody, the coated antibody on the detection line T and the labeled antibody in ② are matched antibodies which respectively form a double anti-sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein matched antibody and the human myeloperoxidase matched antibody
④ and a sample sucking pad closely pressed with the nitrocellulose NC film.
In the above scheme, the preparation method of the vector coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody in a) comprises: and (2) taking a carbonate buffer solution as a solvent, uniformly mixing the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody with the solvent, loading the mixture on a carrier, cleaning the carrier, sealing the carrier by adopting a sealing solution, and drying to obtain the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody.
In the scheme, the carrier is a microporous plate, magnetic particles, a plastic tube or plastic beads.
In the above scheme, the enzyme for labeling in A) is alkaline phosphatase or horseradish peroxidase.
In the above scheme, the chromogenic substrate in the chemiluminescent immunoassay kit of a) is luminol, isoluminol, (adamantane) -1, 2-dioxyethane, 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star; the chromogenic substrate in the chemical chromogenic immunoassay kit is 3,3',5,5' -tetramethyl benzidine or diaminobenzidine.
In the above scheme, the preparation process of the colloidal gold pad in B) is: preparing a colloidal gold solution by a chloroauric acid-trisodium citrate reduction method, adding a marked human eosinophilic granulocyte cationic protein monoclonal antibody and a marked human myeloperoxidase monoclonal antibody into the colloidal gold solution, stirring for 2 hours at room temperature, adding bovine serum albumin with the final concentration of 1 percent and 1 percent polyethylene glycol 20000, sealing for 20 minutes, centrifuging, discarding supernatant, redissolving by using colloidal gold working solution, and uniformly paving the solution on a glass fiber membrane or non-woven fabric to prepare a colloidal gold pad.
The eosinophil cationic protein and myeloperoxidase detection kit is applied to synchronous detection of eosinophil cationic protein and myeloperoxidase products.
The invention has the beneficial effects that: the two kits of the invention adopt a reaction mode of a double-antibody sandwich one-step method, and can simultaneously carry out very specific quantitative and qualitative detection on human eosinophilic granulocyte cationic protein and myeloperoxidase molecules in patient serum and nasal secretion samples. The chemiluminescence immunoassay kit (CLIA) and the chemiluminescence immunoassay kit (ELISA) of the human eosinophil cationic protein and the myeloperoxidase not only effectively utilize the principle of chemiluminescence technology, but also apply enzyme-catalyzed luminescence substrates on the basis of enzyme-linked immunoassay, improve the sensitivity of detection, and simultaneously have the advantages of high stability, good specificity, good accuracy, simple operation and no radioactive pollution. The immunochromatography detection kit (colloidal gold) for the human eosinophil cationic protein and the myeloperoxidase has accurate, stable and reliable detection result; is particularly suitable for the health requirements of the public at the basic level and the vast people in rural areas, and can provide more specific, rapid and reliable basis for the clinical diagnosis of inflammatory diseases such as bronchial asthma, nasosinusitis, rhinitis and the like.
Drawings
FIG. 1 is a diagram showing the results of the immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase in example 2.
FIG. 2 is a line graph of the standards in the kit prepared in example 3.
FIG. 3 shows the correlation between the ECP immunofluorescence assay kit (CLIA) of the present invention and ECP immunofluorescence assay kit (FI) of Thermo Fisher Scientific in the United states, showing that the ECP content in30 samples was measured, and the correlation reached r-0.9757 (P < 0.0001).
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1
A chemiluminescence immunoassay kit (CLIA) and a chemochromic immunoassay kit (ELISA) for human eosinophil cationic protein and myeloperoxidase comprise the following components:
1) human eosinophil cationic protein and myeloperoxidase protein standards;
2) a microplate coated with a monoclonal antibody against human eosinophil cationic protein and a human myeloperoxidase monoclonal antibody;
3) a horseradish peroxidase-labeled human eosinophil cationic protein monoclonal antibody and a myeloperoxidase monoclonal antibody;
4) a chemiluminescent substrate, AMPPD, acted upon by the enzyme;
wherein:
1. the human eosinophil cationic protein and myeloperoxidase standard product is prepared by the following method: mixing 0.02mol/L phosphate buffer solution with pH value of 7.4 and fetal bovine serum according to the volume ratio of 4:1 to prepare a basic buffer solution, and respectively diluting the human eosinophil cationic protein and the myeloperoxidase into the basic buffer solution with the concentrations of 0, 25, 64, 160, 400 and 1000 ng/mL.
2. The preparation method of the microplate coated with the human eosinophil cationic protein monoclonal antibody and the myeloperoxidase monoclonal antibody comprises the following steps:
1) mixing a 50mM carbonate buffer solution with a pH value of 9.6 serving as a solvent with a human eosinophilic granulocyte cationic protein monoclonal antibody and a myeloperoxidase monoclonal antibody to prepare a mixed solution with the concentration of 5 mu g/mL, adding the mixed solution into each hole of a microporous plate, placing the mixed solution at 4 ℃ for 24 hours, wherein each hole is 120 mu L;
2) washing the microplate with PBS (phosphate buffer solution) with pH value of 7.4 for 3 times, wherein each hole is washed with 300 μ L each time;
3) adding 300 mu L of confining liquid with the pH value of 7.3 into each cleaned micropore plate, standing overnight at 4 ℃, throwing off the confining liquid, patting the confining liquid on absorbent paper, dehumidifying and drying at room temperature for 24h, immediately carrying out vacuum packaging, and storing at 4 ℃;
the confining liquid is prepared by the following method: collecting 8g NaCl and 1.43g anhydrous Na2HPO40.24g of anhydrous KH2PO40.2g KCl, 10g BSA, 25g sucrose and 0.5mL Proclin300, addition isolationThe volume of the seed water is 1000 mL.
3. Monoclonal antibodies to alkaline phosphatase-labeled human eosinophil cationic protein and myeloperoxidase
Coupling the human eosinophil cationic protein monoclonal antibody and the monoclonal antibody of myeloperoxidase with horseradish peroxidase by a sodium periodate method, fully dialyzing with PBS buffer solution with the pH value of 7.3, adding glycerol with the same volume into the dialyzed conjugate solution, and diluting with 20% bovine serum enzyme diluent until the concentration of the enzyme-labeled antibody is 1:10000 and preserving at the temperature of-20 ℃;
the formula of the 20% bovine serum enzyme diluent is as follows:
4. the chemiluminescent substrate 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphorus) acted by the enzyme
Acyloxy) phenyl-1, 2-dioxyethane (AMPPD)
The formula of the AMPPD chemiluminescent substrate is as follows:
5. semi-finished product and finished product composition
And subpackaging the products obtained in the steps to obtain semi-finished products. And randomly extracting three parts to carry out specificity, precision, sensitivity and stability test, and assembling the qualified products into a human eosinophilic granulocyte cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and a chemochromic immunoassay kit (ELISA).
Experiments prove that: corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) can be prepared by replacing the microplate of step 2) in example 1 with a plastic tube, and the other steps are the same as example 1.
The magnetic particles or plastic beads are used for replacing the micro-porous plate in the step 2) of the example 1, and a blank micro-porous plate or a plastic tube is put into the kit, and the corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) can be prepared in the same way as the example 1;
experiments prove that: corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) can be prepared as in example 1 by replacing PBS with pH 7.3 or 7.5 or PBST with pH7.4 in step 2 of example 1, or PBST with pH 7.3, 7.4 or 7.5;
experiments prove that: corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) can be prepared by using a blocking solution with pH value of 7.2, 7.4 or 7.5 to replace the blocking solution with pH value of 7.3 in step 2 of example 1, and the method is otherwise the same as example 1;
experiments prove that: corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) can be prepared as in example 1 by substituting (adamantane) -1, 2-dioxyethane, CSPD or CDP-Star for 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane of step 4 of example 1;
experiments prove that: the same procedure as in example 1 was repeated except that the concentrated washing solution having pH 7.3 in step 5 of example 1 was replaced with the concentrated washing solution having pH 7.2, 7.4 or 7.5, to prepare a corresponding human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and a chemiluminescence immunoassay kit (ELISA).
Paired antibody interaction: to avoid non-specific binding between two pairs of partner antibodies, it is necessary to exclude interactions between partner antibodies, and the experimental protocol is designed as follows:
A. presence or absence of cross-reaction between antigen-antibody: mixing a 50mM carbonate buffer solution with a pH value of 9.6 serving as a solvent with human ECP (eosinophilic granulocyte cationic protein) to prepare a mixed solution coated ELISA plate with a concentration of 5 mug/mL, and respectively detecting with a pair of MPO antibodies, wherein the result is negative, and the MPO antibodies can not identify the human ECP (eosinophilic granulocyte cationic protein);
mixing 50mM carbonate buffer solution with the pH value of 9.6 serving as a solvent with human MPO (myeloperoxidase) to prepare a mixed solution coated ELISA plate with the concentration of 5 mug/mL, and detecting by using a pair of ECP antibodies respectively, wherein the result is negative, and the ECP antibodies can not identify the human MPO (myeloperoxidase);
B. presence or absence of cross-reaction between antibody-antibody: mixing 50mM carbonate buffer solution with the pH value of 9.6 serving as a solvent with ECP (eosinophilic cationic protein) and MPO (myeloperoxidase) monoclonal antibodies to prepare a mixed solution coated ELISA plate with the concentration of 5 mug/mL; simultaneously, 50mM carbonate buffer solution with the pH value of 9.6 is adopted as a solvent to be mixed with ECP (eosinophilic granulocyte cationic protein) monoclonal antibody to prepare a mixed solution coated ELISA plate with the concentration of 5 mug/mL; the detection is carried out by ECP protein with the same concentration gradient, and the result shows that: the detection results of the coated single antibody and the coated two antibodies are consistent, which shows that the MPO (myeloperoxidase) monoclonal antibody has no influence on the detection result of ECP;
mixing 50mM carbonate buffer solution with the pH value of 9.6 serving as a solvent with ECP (eosinophilic cationic protein) and MPO (myeloperoxidase) monoclonal antibodies to prepare a mixed solution coated ELISA plate with the concentration of 5 mug/mL; simultaneously, 50mM carbonate buffer solution with the pH value of 9.6 is adopted as a solvent to be mixed with MPO (myeloperoxidase) monoclonal antibody to prepare a mixed solution coated ELISA plate with the concentration of 5 mug/mL; the detection was carried out with MPO (myeloperoxidase) of the same concentration gradient, respectively, and the results showed that: the detection results of the single antibody coated and the two antibodies coated at the same time are consistent, which indicates that the ECP (eosinophilic granulocyte cationic protein) monoclonal antibody has no influence on the detection result of MPO.
A method for detecting a human eosinophil cationic protein and myeloperoxidase using the human eosinophil cationic protein and myeloperoxidase chemiluminescence immunoassay kit (CLIA) and a chemochromic immunoassay kit (ELISA), comprising the steps of:
(1) sample adding: respectively arranging a standard hole, a sample hole to be detected and a blank hole; setting 7 holes of a standard hole, sequentially adding 100 mu L of standard substance with concentration, adding 100 mu L of sample diluent into a blank hole, adding 100 mu L of sample to be detected into the rest hole, and incubating for 30 minutes at 37 ℃ by using an ELISA plate;
(2) discarding the liquid, and spin-drying without washing;
(3) adding 100 mu L of enzyme labeled antibody working solution (prepared before use) into each hole, and incubating for 30 minutes at 37 ℃ by using an enzyme label plate;
(4) discarding liquid in the holes, washing each hole with 250 mu L of washing liquid, soaking for 1 minute, absorbing (not touching the plate wall) or throwing away liquid in the ELISA plate, laying several layers of absorbent paper on a laboratory bench, patting the ELISA plate downwards forcibly for several times (or patting the liquid in the holes to dry), repeatedly washing the plate for 3 times, and completely drying the washing liquid in the holes after the last washing; an automatic plate washing machine is also available.
(5) Add biotin-labeled streptavidin working solution (prepare before using) 100. mu.L per well, incubate 30 minutes at 37 ℃;
(6) discarding liquid in the holes, spin-drying, and washing the plate for 5 times, wherein the method is the same as the step 4;
(7) adding 100 mu L of substrate solution into each hole, and developing the enzyme label plate at 37 ℃ in a dark place (the reaction time is controlled to be 10-15 minutes and is not more than 20 minutes, and the reaction can be stopped when the first 3-4 holes of the standard holes have obvious gradient blue and the second 3-4 holes have no obvious gradient);
(8) adding 50 mu L of termination solution into each hole to terminate the reaction, wherein the blue color is immediately changed into yellow color; the adding sequence of the stop solution is as same as that of the substrate solution as possible, if the color is not uniform, the enzyme label plate is gently shaken to uniformly mix the solution;
(9) immediately after ensuring that there are no water droplets at the bottom of the microplate and no air bubbles in the wells, the optical density (o.d. value) of each well was measured at a wavelength of 450nm with a microplate reader.
Example 2
An immunochromatography detection kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, which is prepared by the following steps:
1. preparing a gold label pad:
human eosinophilic cationic protein and myeloperoxidasePreparing a colloidal gold solution with the diameter of 30-40 nm by using a chemozyme antibody colloidal gold labeled chloroauric acid-trisodium citrate reduction method, and taking three parts of colloidal gold after the preparation is finished, wherein the three parts of colloidal gold are respectively used for preparing 0.2M K2CO3Adjusting the pH of the solution to pH7.5, pH8.0 and pH 8.5; then the solution is placed on a magnetic stirrer to be slowly stirred, 0.5mg, 1mg and 1.5mg of human eosinophil cationic protein and myeloperoxidase antibody are added into the solution per 100ml to be slowly dripped into the colloidal gold solution, the stirring is continued for 2 hours, the solution is added into PEG2000 with the final concentration of 1 percent and BSA with the final concentration of 1 percent to be sealed for 20 minutes, the centrifugation is carried out at 12000r/min after the marking is finished, the supernatant is discarded, and the precipitate is redissolved into colloidal gold working solutions (pH8.0, containing BSA, sheep serum, sucrose and surfactant) with different proportions according to 50 percent of original volume. Then spreading the labeled colloidal gold solution according to 1ml of the solution for 20cm2The mixture is loaded on a glass fiber film or a non-woven fabric, and dried for 2-5 hours in a drying room with the temperature of 20-25 ℃ and the relative humidity of less than 30 percent to prepare the colloidal gold pad.
2. Preparation of a coating film:
NC membrane coating, diluting the coated human eosinophilic granulocyte cationic protein and the myeloperoxidase antibody to 0.5mg/ml, 1mg/ml and 1.5mg/ml by using 0.01M PBS (pH7.4g), respectively diluting the goat anti-mouse IgG to 1mg/ml and 2mg/ml, respectively carrying out streak coating on the NC membrane by using a membrane spraying instrument according to 1.2ul/cm, and drying the NC membrane for 2-5 hours in a drying room with the temperature of 20-25 ℃ and the relative humidity of less than 30 percent after the coating is finished.
3. Assembling a test paper card:
in a drying chamber with the temperature of 20-25 ℃ and the humidity of less than 40%, taking a plastic bottom plate, placing a coated NC film in the middle of the plastic bottom plate for pasting, cutting a colloidal gold pad into a proper width, overlapping the colloidal gold pad on one side of a T line of the NC film, overlapping 1/4 of the colloidal gold pad for pasting, overlapping and pasting a sample-loading pad on the other side of the colloidal gold pad, and overlapping 1/3 of the colloidal gold pad for pasting; overlapping a sample suction pad on one side of the NC film C line, and 1/10 adhering the sample suction pad; and finally, cutting the adhered plastic plate into test strips with the width of 3-5 mm by using a cutting machine, and then putting the test strips into a plastic card to form the test strip card.
Paired antibody interaction: to avoid non-specific binding between two pairs of partner antibodies, it is necessary to exclude interactions between partner antibodies, and the experimental protocol is designed as follows:
A. presence or absence of cross-reaction between antigen-antibody: NC membrane coating human ECP (eosinophilic granulocyte cationic protein) is diluted to 0.5mg/ml, 1mg/ml and 1.5mg/ml by 0.01M PBS (pH7.4), then streaked and coated on the NC membrane by a membrane spraying instrument according to 1.2ul/cm, and after coating is finished, the NC membrane is dried for 2-5 hours in a drying room with the temperature of 20-25 ℃ and the relative humidity of less than 30 percent. The detection result is negative by using a pair of MPO antibodies respectively, and the MPO antibodies can not identify human ECP (eosinophilic granulocyte cationic protein);
NC membrane coating is carried out by diluting human MPO (myeloperoxidase) into 0.5mg/ml, 1mg/ml and 1.5mg/ml by 0.01M PBS (pH7.4), then respectively carrying out streak coating on the NC membrane by a membrane spraying instrument according to 1.2ul/cm, and drying the NC membrane for 2-5 hours in a drying room with the temperature of 20-25 ℃ and the relative humidity of less than 30 percent after coating. Detecting with a pair of ECP antibodies respectively, wherein the result is negative, and the ECP antibodies can not identify human MPO (myeloperoxidase);
B. presence or absence of cross-reaction between antibody-antibody: diluting ECP (eosinophilic granulocyte cationic protein) and MPO (myeloperoxidase) monoclonal antibodies into 0.5mg/ml, 1mg/ml and 1.5mg/ml by using 0.01M PBS (pH7.4PBS) for NC membrane coating, then respectively scribing and coating the NC membrane by using a membrane spraying instrument according to 1.2ul/cm, and drying the NC membrane for 2-5 hours in a drying room with the temperature of 20-25 ℃ and the relative humidity of less than 30 percent after the coating is finished; simultaneously coating ECP (eosinophilic cationic protein) monoclonal antibody by the same process; the detection is carried out by ECP protein with the same concentration gradient, and the result shows that: the detection results of the coated single antibody and the coated two antibodies are consistent, which shows that the MPO (myeloperoxidase) monoclonal antibody has no influence on the detection result of ECP;
diluting ECP (eosinophilic granulocyte cationic protein) and MPO (myeloperoxidase) monoclonal antibodies into 0.5mg/ml, 1mg/ml and 1.5mg/ml by using 0.01M PBS (pH7.4PBS) for NC membrane coating, then respectively scribing and coating the NC membrane by using a membrane spraying instrument according to 1.2ul/cm, and drying the NC membrane for 2-5 hours in a drying room with the temperature of 20-25 ℃ and the relative humidity of less than 30 percent after the coating is finished; coating MPO (myeloperoxidase) monoclonal antibody by the same process; the detection was carried out with MPO (myeloperoxidase) of the same concentration gradient, respectively, and the results showed that: the detection results of the single antibody coated and the two antibodies coated at the same time are consistent, which indicates that the ECP (eosinophilic granulocyte cationic protein) monoclonal antibody has no influence on the detection result of MPO.
The detection method for detecting the human eosinophil cationic protein and the myeloperoxidase by using the immunochromatography detection kit (colloidal gold) of the human eosinophil cationic protein and the myeloperoxidase comprises the following steps:
(1) sampling: the sampling paper strip can be gently placed into nasal cavity (if nasal secretion is more, it can be dipped directly). After the secretion soaks the red line of the paper slip, the part above the red line can be cut off by a pair of small scissors, and then the cut part is put into a secretion soaking bottle and mixed and shaken for about 10 seconds;
(2) sample adding: taking a test pen card, opening a cover in front of the soaking bottle, and dripping 10 drops of soaking liquid into a hole groove at the lower end of the pen card;
(3) color development: the test pen card is vertically placed, so that the liquid can be seen to be soaked upwards and red appears; standing for 15-20 minutes and then reading;
(4) and (4) interpretation of results: reading a mark: the uppermost is the control line (C), the middle is MPO (M), and the lowermost is ECP (E)
1) Result 1 (shown in fig. 1 (a)): if all three lines appear (C, M, E appear), then it can be judged that the infection is dominant, and the anti-infection treatment can be mainly performed in the treatment;
2) result 1 (shown in fig. 1 (b)): if only the upper and lower lines are displayed (C and E are displayed), it can be judged that the anaphylaxis is the main one, and the anti-anaphylaxis treatment can be mainly performed on the treatment;
3) result 1 (shown in fig. 1 (c)): if only the first C line appears, the result is shown by the detection of normal people, and the result can also be shown by other non-infectious and non-allergic rhinitis patients, and the analysis, judgment and treatment are needed according to the specific disease conditions.
Note that ① showed incorrect results in addition to these 3 results and required retesting.
② degree, if M or E is darker than C, it is "+ +", if it is equal to C, it is "+", if it is weaker than C, it is "+", if it is not.
EXAMPLE 3 methodological significance of the kits of the invention
The ECP content in nasal secretion cannot be used as an independent index for diagnosing AR, but the combination of ECP and MPO expression in the nasal secretion can well distinguish AR from CRS. The research aims to analyze inflammatory factors in nasal secretion, so that the nasal inflammation can be more accurately diagnosed, and the nasal secretion is more suitable for clinical diagnosis than nasal secretion cytology. Meanwhile, because the expression of the inflammatory factors of the AR group and the CRS group is obviously higher than that of the healthy control group, the ECP and MPO levels are reduced and close to normal after treatment, and the inflammatory factors can be expected to become objective indexes in the treatment of rhinitis and nasosinusitis.
As shown in fig. 2(a), ECP concentrations were significantly increased in nasal secretions of AR group and CRS group compared to the control group (arv. s control 0.0008; AR v.s CRS P <0.0001), and CRS group was significantly higher than AR group (P0.0056); compared with the AR group and the control group, the nasal secretion of the CRS group had a significant increase in MPO concentration (both P <0.0001), and the AR group was slightly higher than the control group (P ═ 0.3105).
As shown in fig. 2(b), the percentage of eosinophils in the cytological examination of AR group was significantly higher than in CRS group and control group (P <0.0001), with CRS group slightly higher than control group (P ═ 0.0493); the percentage of neutrophils in the cytological examination of the CRS group was significantly higher than that of the AR group and the control group (P <0.0001), and the AR group was slightly higher than that of the control group (P ═ 0.0163). (see FIG. 2).
Example 4
The clinical application of the chemiluminescence immunoassay kit (CLIA) of the human eosinophil cationic protein and myeloperoxidase is compared with ECP immunofluorescence assay kit of Thermo Fisher Scientific company in the United states:
32 patients with allergic rhinitis and 30 patients with chronic sinusitis were simultaneously tested using the kit prepared in example 1 of the present invention and ECP immunofluorescence assay kit of Thermo Fisher Scientific Inc. USA, and the correlation between the two kits was compared.
First, source and inclusion criteria for clinical serum specimens:
32 allergic rhinitis patients, 18 males and 14 females, age 4-41 years, average (19.9 +/-10.8) years old, standard reference allergic rhinitis diagnosis and treatment guidelines (2015, Tianjin) are included, ① symptoms of sneezing, watery nasal discharge, nasal itching, nasal obstruction and the like appear in the symptoms of ① symptoms, the symptoms continue or accumulate for more than 1 hour each day and can be accompanied by eye symptoms of itching, lacrimation, redness of eyes and the like, ② signs of common nasal mucosa pallor, edema and nasal water sample secretion, ③ allergen detection, at least one allergen SPT and/or serum specific IgE is positive.
30 patients with chronic rhinosinusitis, 16 men, 13 women, age 3-28 years old, and average (9.2 +/-7.3) years old are brought into standard reference chronic rhinosinusitis diagnosis and treatment guidelines (2012, Kunming): ① symptoms of nasal obstruction, viscosity or purulent rhinorrhea are required to be one of nasal obstruction, viscosity or purulent rhinorrhea and can be combined with head and face distending pain, hyposmia or loss, ② nasal endoscopy examination of viscous or purulent secretion from inner nasal passages and olfaction, nasal mucosa congestion, edema and uncomplicated polyp, ③ image examination of sinus CT scanning shows inflammatory lesion of sinus oral nasal passage complex and/or nasal sinus mucosa, and nasosinusitis selected in the research is not combined with allergic rhinitis in order to reduce allergic factor interference.
21 normal persons, 2 men, 19 women, age 32-60 years, mean (38.3 ± 5.6) years, inclusion criteria: has no history of rhinitis and has no respiratory tract diseases recently.
This experiment was approved by the ethical committee of the university of science and technology in china.
Secondly, the nasal secretion samples are respectively detected by using the kit of the embodiment 1 and ECP immunofluorescence assay kit (operated according to the instruction) of the Thermo Fisher Scientific company of the United states, and the results are statistically analyzed and processed, which shows that the detection results of the two methods are highly correlated, and are shown in the attached figure 3.
Three, result in
Comparative data using a chemiluminescent immunoassay kit (CLIA) using human eosinophil cationic protein and myeloperoxidase and ECP immunofluorescence assay kit from Thermo Fisher Scientific, USA, for the same serum samples show that the two methods are highly correlated, with a correlation coefficient r > 0.98 and a P value of less than 0.01. The two methods have equal use value.
It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.
Claims (7)
1. A human eosinophil cationic protein and myeloperoxidase detection kit is characterized by comprising the following two types:
A) chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) of human eosinophil cationic protein and myeloperoxidase, which comprises the following components:
① human eosinophil cationic protein and human myeloperoxidase standard;
② a vector coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody;
③ labeled human eosinophil cationic protein monoclonal antibody and human myeloperoxidase monoclonal antibody, ③ labeled antibody and ② coating antibody are paired antibodies which respectively form a double-anti-sandwich structure with human eosinophil cationic protein and human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein paired antibody and the human myeloperoxidase paired antibody;
④ substrate for color development;
B) an immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, comprising:
① sample loading pad adhered to one end of the bottom plate;
② a colloidal gold pad containing a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody, which is tightly pressed against the loading pad;
③ nitrocellulose NC membrane tightly pressed with one end of the colloidal gold pad, the nitrocellulose NC membrane is coated with a detection line T and a quality control line C which are separated from each other, the detection line T is coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody which are matched with the ② labeled antibody, the quality control line C is coated with a goat anti-mouse IgG antibody, the coated antibody on the detection line T and the labeled antibody in ② are matched antibodies which respectively form a double anti-sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein matched antibody and the human myeloperoxidase matched antibody
④ and a sample sucking pad closely pressed with the nitrocellulose NC film.
2. The kit for detecting cationic protein and myeloperoxidase of human eosinophils according to claim 1, wherein the preparation method of the vector coated with monoclonal antibody against cationic protein and monoclonal antibody against human eosinophils of A) comprises: and (2) taking a carbonate buffer solution as a solvent, uniformly mixing the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody with the solvent, loading the mixture on a carrier, cleaning the carrier, sealing the carrier by adopting a sealing solution, and drying to obtain the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody.
3. The kit of claim 1, wherein said carrier is a microplate, magnetic particles, plastic tubes or plastic beads.
4. The kit for detecting cationic protein and myeloperoxidase of human eosinophils according to claim 1, wherein said enzyme for labeling in A) is alkaline phosphatase or horseradish peroxidase.
5. The human eosinophil cationic protein and myeloperoxidase detection kit of claim 1, wherein the chromogenic substrate in said chemiluminescent immunoassay kit of A) is luminol, isoluminol, (adamantane) -1, 2-dioxyethane, 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star; the chromogenic substrate in the chemical chromogenic immunoassay kit is 3,3',5,5' -tetramethyl benzidine or diaminobenzidine.
6. The kit for detecting cationic protein and myeloperoxidase of human eosinophils according to claim 1, wherein said colloidal gold pad of B) is prepared by the following steps: preparing a colloidal gold solution by a chloroauric acid-trisodium citrate reduction method, adding a marked human eosinophilic granulocyte cationic protein monoclonal antibody and a marked human myeloperoxidase monoclonal antibody into the colloidal gold solution, stirring for 2 hours at room temperature, adding bovine serum albumin with the final concentration of 1 percent and 1 percent polyethylene glycol 20000, sealing for 20 minutes, centrifuging, discarding supernatant, redissolving by using colloidal gold working solution, and uniformly paving the solution on a glass fiber membrane or non-woven fabric to prepare a colloidal gold pad.
7. Use of the eosinophil cationic protein and myeloperoxidase detection kit of any of claims 1 ~ 6 for the simultaneous detection of eosinophil cationic protein and myeloperoxidase products.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080090252A1 (en) * | 2006-08-30 | 2008-04-17 | Jens Ponikau | Detecting a bacterial process in chronic rhinosinusitis |
| CN101377501A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Cell keratin 19 fragments chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN102192976A (en) * | 2010-03-05 | 2011-09-21 | 复旦大学附属华山医院 | Method for examining and analyzing phlegm, and purpose thereof |
| CN103033624A (en) * | 2012-12-16 | 2013-04-10 | 天津市协和医药科技集团有限公司 | Human myeloperoxidase chemiluminescent immunodetection kit |
| CN104122395A (en) * | 2013-04-28 | 2014-10-29 | 北京协和洛克生物技术有限责任公司 | Kit for detecting myeloperoxidase (MPO) concentration in sample and preparation method of kit |
| CN204330777U (en) * | 2014-05-19 | 2015-05-13 | 江苏金标世纪生物科技有限公司 | People's myeloperoxidase (MPO) fast quantification immunochromatographytest test kit |
| CN204359796U (en) * | 2015-01-05 | 2015-05-27 | 陈建军 | A kind of test strips detecting ECP and MPO |
| CN104950111A (en) * | 2015-05-22 | 2015-09-30 | 北京协和洛克生物技术有限责任公司 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
| CN108445215A (en) * | 2018-02-01 | 2018-08-24 | 浙江艾明德生物科技有限公司 | A kind of kit and preparation method quantitatively detecting myeloperoxidase |
-
2019
- 2019-08-14 CN CN201910749882.4A patent/CN110716052B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080090252A1 (en) * | 2006-08-30 | 2008-04-17 | Jens Ponikau | Detecting a bacterial process in chronic rhinosinusitis |
| CN101377501A (en) * | 2007-08-31 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Cell keratin 19 fragments chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN102192976A (en) * | 2010-03-05 | 2011-09-21 | 复旦大学附属华山医院 | Method for examining and analyzing phlegm, and purpose thereof |
| CN103033624A (en) * | 2012-12-16 | 2013-04-10 | 天津市协和医药科技集团有限公司 | Human myeloperoxidase chemiluminescent immunodetection kit |
| CN104122395A (en) * | 2013-04-28 | 2014-10-29 | 北京协和洛克生物技术有限责任公司 | Kit for detecting myeloperoxidase (MPO) concentration in sample and preparation method of kit |
| CN204330777U (en) * | 2014-05-19 | 2015-05-13 | 江苏金标世纪生物科技有限公司 | People's myeloperoxidase (MPO) fast quantification immunochromatographytest test kit |
| CN204359796U (en) * | 2015-01-05 | 2015-05-27 | 陈建军 | A kind of test strips detecting ECP and MPO |
| CN104950111A (en) * | 2015-05-22 | 2015-09-30 | 北京协和洛克生物技术有限责任公司 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
| CN108445215A (en) * | 2018-02-01 | 2018-08-24 | 浙江艾明德生物科技有限公司 | A kind of kit and preparation method quantitatively detecting myeloperoxidase |
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