AU2021104254A4 - Fluorescence immunochromatography assay test strip for detecting eosinophil cationic protein, use thereof, and detection method - Google Patents
Fluorescence immunochromatography assay test strip for detecting eosinophil cationic protein, use thereof, and detection method Download PDFInfo
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Abstract
OF THE DISCLOSURE
The present disclosure provides a fluorescence immunochromatography assay (FICA) test strip
for detecting eosinophil cationic protein (ECP), use thereof, and a detection method, and relates to
the technical field of FICA techniques. The FICA test strip of the present disclosure includes a bottom
plate, and a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper
that are sequentially lapped on the bottom plate, where the conjugate pad is coated with a first anti
ECP antibody labeled with fluorescent microspheres, the NC membrane is coated sequentially with a
second anti-ECP antibody (T line) and an anti-mouse IgG antibody (C line), and the T line is a test
line and the C line is a control line. The present disclosure allows accurate and quantitative detection
of ECP content in a sample and is used for auxiliary diagnosis of allergic diseases such as allergic
asthma, having the advantages of simple operation, short detection time, high accuracy, and high
sensitivity.
1/1
Conjugate pad C line
Sample pa
Absorbent paper
Bottom plate NC membrane
T line
FIG. 1
Data: Datal_B
3.5 - Model: Logistic
Equation: y = A2 + (Al-A2)/(1 + (x/x0)Ap)
Weighting:
3.0- y No weighting
ChiA2/DoF = 0.00286
RA2 = 0.99915
2.5
Al 0.27409 ±0.08533
A2 407479 ±026345
- 2.0 x0 50.39264 ±6.75284
p 108522 ±0.13173
1.0
0
U)
0.5
0 .0 , , , , , i , , , , , i
1 10 100
Log 1o (ECP standard concentration, ng/mL)
FIG. 2
Description
1/1
Conjugate pad C line Sample pa Absorbent paper
Bottom plate NC membrane T line
FIG. 1
Data: Datal_B 3.5 - Model: Logistic Equation: y = A2 + (Al-A2)/(1 + (x/x0)Ap) Weighting: y No weighting 3.0- ChiA2/DoF = 0.00286 RA2 = 0.99915 2.5 Al 0.27409 ±0.08533 A2 407479 ±026345 - 2.0 x0 50.39264 ±6.75284 p 108522 ±0.13173
0 1.0 U)
0.5
0 .0 , , , , , i , , , , , i 1 10 100
Log 1 o(ECP standard concentration, ng/mL)
FIG. 2
[01] The present disclosure belongs to the technical field of fluorescence immunochromatography assay (FICA) test strips, and specifically relates to an FICA test strip for detecting eosinophil cationic protein (ECP), use thereof, and a detection method.
[02] Bronchial asthma (BA) is a chronic respiratory disease (CRD) that seriously jeopardizes human health, which is the most common airway inflammatory disease in children. There are about 300 million people suffering from BA worldwide. Results of the epidemiological survey nationwide show that the incidence of childhood asthma is increasing year by year, which is consistent with the generally-rising prevalence of BA worldwide. Eosinophil cationic protein (ECP) is a strongly-basic granule protein released by activated eosinophil (EOS), which has extremely high cytotoxicity. ECP is a specific marker of activated EOS and can reflect the activation degree of EOS. Recently, it has been found that ECP can cause airway hyperresponsiveness (AHR) or BA attack and aggravate the allergic inflammatory process.
[03] Since Paul Erhich first described the histochemical properties of EOS in 1879, more and more studies have shown that there are EOS and granule protein released thereby in the serum, urine, tears, and nasal and bronchial secretions of patients with allergic diseases such as asthma, rhinitis, atopic dermatitis, and allergic conjunctivitis. In 1906, Clemens proposed the concept of allergy which was defined as follows: "a specific acquired response occurring in an organism after initial exposure to a foreign protein", which is characterized by the local aggregation and activation of EOS. EOS, after being activated, mainly secretes 4 strongly basic granule proteins: major basic protein (MBP), ECP, EOS-derived neurotoxin, and EOS peroxidase, where ECP is an EOS activity marker and has been widely used in the diagnosis, treatment, and curative effect determination for allergic diseases. It can be known that ECP detection has gradually become one of the detection criteria for allergic diseases, allowing a doctor to clinically identify the applicability of a treatment plan to a patient based on the change in ECP content of the patient before and after treatment.
[04] However, there are few products for detecting ECP in a sample, and a detection method is substantially enzyme-linked immunosorbent assay (ELISA), which is time-consuming. For example, the ImmunoCAPTM ECP widely used abroad involves fluorescent ELISA, which takes four reaction steps and more than two hours to complete a single test.
[05] Preferred embodiments of the present invention seek to provide a fluorescence immunochromatography assay (FICA) test strip for detecting ECP and use thereof, which can quantitatively detect ECP content in a blood sample. The FICA test strip of the present disclosure has the advantages of rapid ECP quantification (a result can be read 15 min after sample addition), high detection accuracy, and high sensitivity.
[06] The present disclosure provides the following technical solutions.
[07] The present disclosure provides an FICA test strip for detecting ECP, including a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent paper that are sequentially lapped on the bottom plate, where the conjugate pad is coated with a first anti-ECP antibody labeled with fluorescent microspheres;
[08] the NC membrane includes a test line and a control line, the test line is coated with a second anti-ECP antibody, and the control line is coated with an anti-mouse IgG antibody; and
[09] the first anti-ECP antibody labeled with fluorescent microspheres is paired with the second anti-ECP antibody.
[10] Preferably, the fluorescent microspheres have a particle size of 50 nm to 500 nm.
[11] Preferably, the first anti-ECP antibody labeled with fluorescent microspheres is coated on the conjugate pad at a concentration of 2 mg/mL to 10 mg/mL, the anti-mouse IgG antibody is coated on the control line at a concentration of 0.5 mg/mL to 5 mg/mL, and the second anti-ECP antibody is coated on the test line at a concentration of 1 mg/mL to 5 mg/mL.
[12] The present disclosure also provides the use of the FICA test strip according to the above technical solution in the preparation of a kit for detecting ECP content in a sample.
[13] The present disclosure also provides a method for detecting ECP content in a sample, including the following steps: adding 70 L to 120 tL of blood sample solution dropwise on a sample pad of the FICA test strip according to the above technical solution, reading a fluorescence signal ratio on the FICA test strip 10 min to 15 min later, and determining ECP content according to a standard curve;
[14] the standard curve is:
[15] y = 4.07479 + (0.27409 - 4.07479)/(1 + (x/50.39264) 1.08522); R2 = 0.99915;
[16] where x is the ECP concentration in the sample solution, with a unit of ng/mL, and y is the fluorescence signal ratio.
[17] The present disclosure provides an FICA test strip for detecting ECP, including a bottom plate, and a sample pad, a conjugate pad, an NC membrane, and an absorbent paper that are sequentially lapped on the bottom plate. The conjugate pad is coated with a first anti-ECP antibody labeled with fluorescent microspheres, the NC membrane is coated sequentially with a second anti-ECP antibody (T line) and an anti-mouse IgG antibody (C line), and the T line is a test line and the C line is a control line. In the present disclosure, the first anti-ECP antibody labeled with fluorescent microspheres is paired with the second anti-ECP antibody coated on the NC membrane; and the first anti-ECP antibody labeled with fluorescent microspheres serves as a secondary antibody, and the second anti ECP antibody coated on the NC membrane serves as a primary antibody. The present disclosure can accurately and quantitatively detect ECP content in a sample, which has the advantages of simple operation (only one step of sample addition is required), rapid ECP quantification (a result can be read 15 min after sample addition), low cost (only a dry fluorescence reader is required as a supporting instrument, with a price of < 3,000 Chinese Yuan; and reagent cost: < 5 Chinese Yuan/person), high detection accuracy, and high sensitivity (< 0.2 ng/mL).
[18] FIG. 1 is a structural diagram of the FICA test strip of the present disclosure; and
[19] FIG. 2 shows a standard curve for FICA.
[20] The present disclosure provides an FICA test strip for detecting eosinophil cationic protein (ECP), including a bottom plate, and a sample pad, a conjugate pad, an NC membrane, and an absorbent paper that are sequentially lapped on the bottom plate, where the conjugate pad is coated with a first anti-ECP antibody labeled with fluorescent microspheres;
[21] the NC membrane includes a test line and a control line, the test line is coated with a second anti-ECP antibody, and the control line is coated with an anti-mouse IgG antibody; and
[22] the first anti-ECP antibody labeled with fluorescent microspheres is paired with the second anti-ECP antibody.
[23] The FICA test strip of the present disclosure preferably has a structure shown in FIG. 1, where a sample pad, a conjugate pad, an NC membrane, and an absorbent paper are sequentially lapped on a bottom plate.
[24] In the present disclosure, the bottom plate preferably includes a PVC bottom plate.
[25] In the present disclosure, the fluorescent microspheres may have a particle size preferably of nm to 500 nm, more preferably of 100 nm to 300 nm, and most preferably of 200 nm.
[26] In the present disclosure, the fluorescent microspheres preferably include inorganic/organic fluorescent microspheres, organic/inorganic fluorescent microspheres, inorganic/inorganic fluorescent microspheres, or organic/organic fluorescent microspheres. In the present disclosure, for the inorganic/organic fluorescent microspheres, an inorganic material is used as a carrier and an organic compound is used as a fluorescent substance; for the organic/inorganic fluorescent microspheres, an organic material is used as a carrier and an inorganic compound is used as a fluorescent substance; for the inorganic/inorganic fluorescent microspheres, an inorganic material is used as a carrier and an inorganic fluorescent nanocrystal with active groups is used as a fluorescent substance; and the organic/organic fluorescent microspheres are polymer fluorescent microspheres formed by polymerization of an organic fluorescent substance and an organic material carrier.
[27] In the present disclosure, the fluorescent microspheres are preferably time-resolved fluorescent microspheres. In specific implementation of the present disclosure, the fluorescent microspheres is MD033 (Item No.) purchased from Nanjing Microdetection Bio-Tech Co., Ltd.
[28] In the present disclosure, the first anti-ECP antibody is preferably a biotinylated anti-ECP antibody, and the first anti-ECP antibody is preferably obtained by mixing biotin with an anti-ECP antibody, where a volume ratio of the biotin to the anti-ECP antibody is preferably 1:20. In the present disclosure, the anti-ECP antibody is preferably KL8615R (Item No.) purchased from Shanghai Kanglang Biotechnology Co., Ltd.
[29] In the present disclosure, a method for coating the first anti-ECP antibody labeled with fluorescent microspheres on the conjugate pad may preferably include the following steps:
[30] (a) mixing fluorescent microspheres with a first anti-ECP antibody to obtain a first anti-ECP antibody labeled with fluorescent microspheres; and
[31] (b) spray-coating the first anti-ECP antibody labeled with fluorescent microspheres on the conjugate pad.
[32] In the present disclosure, fluorescent microspheres are mixed with a first anti-ECP antibody to obtain the first anti-ECP antibody labeled with fluorescent microspheres first. In the present disclosure, the volume ratio of the fluorescent microspheres to the first anti-ECP antibody is preferably 10:1.
[33] In the present disclosure, after the first anti-ECP antibody labeled with fluorescent microspheres is obtained, the first anti-ECP antibody labeled with fluorescent microspheres is spray coated on the conjugate pad. In the present disclosure, the first anti-ECP antibody labeled with fluorescent microspheres may be spray-coated on the conjugate pad at an amount preferably of 2 pL/cm to 10 L/cm and more preferably of 5 L/cm to 8 L/cm. In the present disclosure, the first anti-ECP antibody labeled with fluorescent microspheres serves as a secondary antibody.
[34] In the present disclosure, the control line and test line on the NC membrane are preferably arranged in parallel; there are preferably one test line and one control line; the test line (T line) is coated with a second anti-ECP antibody, and the second anti-ECP antibody may be coated at a concentration preferably of 0.5 mg/mL to 10 mg/mL, more preferably of 1 mg/mL to 8 mg/mL, and most preferably of 3 mg/mL to 5 mg/mL, and the control line (C line) is coated with an anti-mouse IgG antibody, and the anti-mouse IgG antibody may be coated at a concentration preferably of 0.5 mg/mL to 5 mg/mL and more preferably of 1 mg/mL to 3 mg/mL. In the present disclosure, the second anti-ECP antibody coated on the test line on the NC membrane serves as a primary antibody, which is paired with the first anti-ECP antibody labeled with fluorescent microspheres (secondary antibody). A
[35] In the present disclosure ,there is no special limitations on a preparation method of the NC membrane, and the preparation method preferably include: diluting the second anti-ECP antibody and anti-mouse IgG antibody with a 20 mM Tris-HCl buffer (pH 9.0, a coating buffer), separately; and streaking the obtained two diluted antibody solutions on an NC membrane in parallel, separately. When the antibodies penetrate into the NC membrane, a test zone coated with the second anti-ECP antibody and a control zone coated with the anti-mouse IgG antibody are formed. In the present disclosure, the test zone is preferably streaked at a diluted antibody concentration of 3 mg/ml (liquid output of peristaltic pump: 0.5 mL/cm, and streaking speed: 50 m/20 min) and blast-dried in a drying oven at 37°C for 12 h; and the control zone is preferably streaked on the NC membrane at a diluted antibody concentration of 5 mg/ml (liquid output of peristaltic pump: 0.5 mL/cm, and streaking speed: m/20 min) and blast-dried in a drying oven at 37°C for 12 h, which is parallel to the test zone. After the streaking, the present disclosure preferably further include: blocking the NC membrane at 37°C for 60 min with a blocking solution, drying the membrane at 37°C for 2 h, and packaging for later use.
[36] In specific implementation of the present disclosure, the second anti-ECP antibody is preferably ABP51222 (Item No.) purchased from Wuhan AmyJet Scientific Inc.
[37] In the present disclosure, the sample pad, the conjugate pad, the NC membrane, and the absorbent filter paper are preferably assembled and adhered on the PVC bottom plate sequentially, and cut into test strips shown in FIG. 1 as required (4 mm) with a strip cutter.
[38] The FICA test strip of the present disclosure can quantitatively detect ECP content in a blood sample. A doctor can identify the applicability of a treatment plan to a patient based on the change in ECP content of the patient before and after treatment.
[39] The present disclosure also provides the use of the FICA test strip described above in the detection of ECP content in a sample.
[40] The present disclosure also provides a method for detecting ECP content in a sample, including the following steps: adding 70 L to 120 L of a sample solution dropwise on a sample pad of the FICA test strip described above, reading a fluorescence signal ratio on the FICA test strip 10 min to 15 min later, and calculating ECP content according to a standard curve.
[41] The standard curve is:
[42] y = 4.07479 + (0.27409 - 4.07479)/(1 + (x/50.39264) 1.08522); R2 = 0.99915;
[43] where x is an ECP concentration in a sample solution (ng/mL), and y is a fluorescence signal ratio (T/C).
[44] In the present disclosure, 70 [ to 120 L of a target analyte is added dropwise on the sample pad, and after reaction in the control zone is completed 10 min to 15 min later, the test strip is placed in a special fluorescence signal reader to read a fluorescence signal ratio, thus achieving quantitative determination. In the present disclosure, after being added dropwise on the sample pad, the target 1Z analyte binds to the first anti-ECP antibody labeled with fluorescent microspheres on the conjugate pad, then reacts with the second anti-ECP antibody on the test zone, and finally reaches the control zone where the test is completed.
[45] The FICA test strip for detecting ECP, use thereof, and detection method provided in the present disclosure will be described in detail below with reference to examples, but these examples should not be understood as limiting the claimed scope of the present disclosure.
[46] Example 1
[47] 1. Preparation of conjugate pad
[48] 1) Preparation of a biotinylated anti-ECP antibody
[49] An anti-ECP antibody (Shanghai Kanglang Biotechnology Co., Ltd., Item No.: KL8615R) was subjected to adequate dialysis with 10 mM PBS (pH 7.4), 1 mL of NHSB was dissolved with 1 mL of dimethyl sulfoxide (DMSO) to obtain an NHSB solution, 25 L of the NHSB solution was added to the anti-ECP antibody, and the resulting mixture was stirred for 2 h to 4 h and then further stirred at room temperature for 10 min, and then subjected to dialysis with 10 mM PBS (pH 7.4) to obtain the biotinylated anti-ECP antibody.
[50] 2) Preparation of anti-ECP antibody labeled with fluorescent microspheres
[51] Fluorescent microspheres (Nanjing Microdetection Bio-Tech Co., Ltd., MD033) were mixed with the biotinylated antibody prepared in step 1) at a volume ratio of 10:1; after a reaction was conducted for 30 min, the resulting reaction solution was centrifuged; and the resulting precipitate was dissolved in a storage buffer and diluted to the original volume.
[52] 3) The anti-ECP antibody labeled with fluorescent microspheres was spray-coated on the conjugate pad at an amount of 2 l/cm to 10 l/cm.
[53] 2. Preparation of NC membrane
[54] 1) Membrane treatment: A piece of NC membrane was taken, marked, and soaked in a membrane treatment solution (TBS, pH 7.4) for 5 min to 10 min.
[55] 2) Assembly of spotting device: The soaked NC membrane was placed on a flat mat, and an antibody spotting board was placed, leaving a place for attaching marker paper.
[56] 3) Preparation of anti-ECP antibody test zone: The anti-ECP antibody (Wuhan AmyJet Scientific Inc., Item No.: ABP51222) was streaked at a concentration of 3 mg/ml (liquid output of peristaltic pump: 0.5 mL/cm, and streaking speed: 50 m/20 min) and blast-dried in a drying oven at 37°C for 12 h.
[57] 4) Preparation of control zone: The anti-mouse IgG antibody (Shenzhen Otwo Biotech Inc., Item No.: HT1620093) was streaked on the NC membrane at a concentration of 3 mg/mL (liquid output of peristaltic pump: 0.5 mL/min, and streaking speed: 50 m/20 min) and blast-dried in a drying oven at 37°C for 12 h, which was parallel to the test zone.
[58] 5) The NC membrane was blocked at 37°C for 60 min with a blocking solution (prepared from 100 mL of PBS and 0.5 g of BSA), dried at 37°C for 2 h, and packaged for later use.
[59] 3. Assembly of test strip
[60] The sample pad, the conjugate pad, the NC membrane, and the absorbent filter paper were sequentially assembled and adhered on a PVC bottom plate, and the resulting product was cut into test strips shown in FIG. 1 as required (4 mm) with a strip cutter.
[61] Example 2
[62] 1. The assembled test strips in Example 1 were put into upper and lower part of a plastic case for detecting an antigen to be tested:
[63] 100 1 of target analyte was added dropwise on the sample pad, which bound to the anti-ECP antibody labeled with fluorescent microspheres on the conjugate pad, then reacted with the anti-ECP antibody on the test zone, and finally reached the control zone where the test was completed. Then the test strip was placed in a special fluorescence signal reader to read a fluorescence signal ratio, thus achieving quantitative determination.
[64] 2. Linearity of dosage-response curve: Calibration solution series prepared from ECP calibrators were detected, with concentrations of 200.00 ng/mL, 100.00 ng/mL, 50 ng/mL, 25 ng/mL, ng/mL, and 2 ng/mL, and the S curve was used for fitting. Results show that, within a concentration range of 2 ng/mL to 200 ng/mL, a relationship between concentrations and test values presents a smooth increasing curve, and the equation for the curve is shown in FIG. 2:
[65] y = 4.07479 + (0.27409-4.07479)/(1 + (x/50.39264) 10. 8 5 2 2 ), R2 = 0.99915. According to the standard equation, there is an extremely high correlation between the ECP concentration value and the fluorescence signal ratio (T/C).
[66] 3. Detection of positive samples and negative control samples
[67] 100 L of target analyte (from the Allergy Department of a First-Class Hospital at Grade 3) dropwise on the sample pad, and timing was immediately started with a timer, and 10 min to 15 min later, a result was read using a dry fluorescence immunoassay analyzer (Guangzhou Lanbo Biological Technology Co., Ltd., AFS-1000).
[68] ImmunoCAPTMECP (affiliated to the brand of phadia, Thermofisher) widely used worldwide in the art involves the methodology of fluorescent ELISA, where four reaction steps and more than two hours are required to complete a single test, and dedicated automation equipment must be used to complete a test, resulting in an automation equipment investment of > 600,000 Chinese Yuan and a reagent cost of > 80 Chinese Yuan/person.
[69] The comparison between the detection result of the present disclosure and the detection result of ImmunoCAPTM ECP is shown in Table 1.
[70] Table 1 Detection results of positive samples and negative control samples Productofthepresent ImmunoCAPTM ECP Domestic ELISA kit disclosure No. Sample type result positive/ result positive/n result positive/neg (ng/mL) negative (ng/mL) egative (ng/mL) ative Positive 1 19.2 + 22.6 + 1985.9
+ sample 1 Positive 2 45.1 + 53.8 + 1982.2
+ sample 2 Positive 3 33.6 + 34.2 + 306.5
+ sample 3 Positive 4 57.3 + 55.7 + 1107.1
+ sample 4 Positive 5 52.8 + 52.4 + 653.8
+ sample 5 Positive 6 47.5 + 65.3 + 579.3
+ sample 6 Positive 7 61.6 + 60.7 + 1260.1
+ sample 7
8 Healthy 11.8 - 12.4 - 1222.5
+ control 1
9 Healthy 6.9 - 6.7 - 1719.0 + control 2
10 Healthy 10.2 - 9.9 - 884.7 +
control 3
[71] The results in Table 1 show that the domestic ELISA kit shows false positives for all healthy controls, indicating poor specificity. Detection results of the present disclosure are 100% consistent with that of the imported fluorescent ELISA reagent for both the positive and negative samples, indicating that the present disclosure can achieve the same clinical performance as the imported fluorescent ELISA reagent, with shorter detection time, lower cost, excellent specificity, and high sensitivity.
[72] The above descriptions are merely preferred embodiments of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.
[731 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
[74] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Claims (5)
- WHAT IS CLAIMED IS: 1. A fluorescence immunochromatography assay test strip for detecting eosinophil cationic protein (ECP), comprising a bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent paper that are sequentially lapped on the bottom plate, wherein: the conjugate pad is coated with a first anti-ECP antibody labeled with fluorescent microspheres; the NC membrane comprises a test line and a control line, the test line is coated with a second anti-ECP antibody, and the control line is coated with an anti-mouse IgG antibody; and the first anti-ECP antibody labeled with fluorescent microspheres is paired with the second anti ECP antibody.
- 2. The fluorescence immunochromatography assay test strip according to claim 1, wherein the fluorescent microspheres have a particle size of 50 nm to 500 nm.
- 3. The fluorescence immunochromatography assay test strip according to claim 1, wherein the first anti-ECP antibody labeled with fluorescent microspheres is coated on the conjugate pad at a concentration of 2 mg/mL to 10 mg/mL, the anti-mouse IgG antibody is coated on the control line at a concentration of 0.5 mg/mL to 5 mg/mL, and the second anti-ECP antibody is coated on the test line at a concentration of 1 mg/mL to 5 mg/mL.
- 4. Use of the fluorescence immunochromatography assay test strip according to any one of claims 1 to 3 in the preparation of a kit for detecting ECP content in a sample.
- 5. A method for detecting ECP content in a sample, comprising the following steps: adding 70 pL to 120 L of blood sample solution dropwise on a sample pad of the FICA test strip according to any one of claims 1 to 3, reading a fluorescence signal ratio on the FICA test strip 10 min to 15 min later, and determining ECP content according to a standard curve; wherein the standard curve is: y = 4.07479 + (0.27409 - 4.07479)/(1 + (x/50.39264) 1.08522); R2 = 0.99915; wherein x is the ECP concentration in the blood sample solution in a unit of ng/mL, and y is the fluorescence signal ratio.
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