CN115184621A - Copeptin detection reagent based on immunolayer analysis and preparation method thereof - Google Patents

Abstract

The present invention provides a copeptin detection reagent based on immune layer analysis, including polystyrene nano-fluorescent microspheres, a solution of colored polystyrene microspheres and an anti- and peptidin antibody coupling solution, and a chromatographic solution. The liquid volume percentage is 0.01%-1% Tween-20, the mass concentration is 0.1%-1.5% BSA and 1.5%-2.2% sucrose, and the solvent is a molar concentration of 0.015mol/l-0.035mol/l and a pH of 7.0- Phosphate buffer solution according to 7.4; the present invention also provides a preparation method of copeptin detection reagent based on immunolayer analysis, S1. Dilute carboxyl polystyrene nano-fluorescent microspheres and colored polystyrene microspheres with labeling buffer To the solid content of about 0.2%, add EDC solution and Sulfo-NHS-LC-Biontin biotin labeling test solvent, etc., using immunochromatography technology, the specific binding reaction of the two antibodies to form a specific "antibody-antigen-antibody" immune system The complex is combined with a fluorescent or colored tracer label at the same time, and the reagent card and the fluorometer can be used for quantitative or qualitative detection of capeptin in human blood samples.

Description

Copeptin detection reagent based on immunolayer analysis and preparation method thereof

technical field

The invention belongs to the technical field of immunology, and more particularly, relates to an immunolayer analysis-based copeptin detection reagent; in particular, it relates to a preparation method of an immune layer analysis-based copeptin detection reagent.

Background technique

With the advent of an aging population, the incidence of cardiovascular and cerebrovascular diseases is increasing, and the mortality and disability rates are very high. Cardiovascular and cerebrovascular infarction is the number one cause of human death worldwide. The main method to reduce the incidence of cardiovascular and cerebrovascular infarction is early prevention, early diagnosis and early treatment. Copeptin (CPP) is a polypeptide containing 39 amino acid residues homologous to arginine vasopressin (AVP) and is the C-terminal part of pro-arginine vasopressin (pro-VAP). Peptides. Studies have found that detecting the level of Copeptin (CPP) in the blood can early diagnose the occurrence of cardiovascular and cerebrovascular infarction, and it is necessary to vigorously develop a kit for detecting CPP in the blood.

Arginine vasopressin (AVP) is a short peptide containing 9 amino acids formed by the processing and cleavage of pro-arginine vasopressin (Pre-provasopressin), also known as antidiuretic hormone or vasopressin. Studies have found that AVP can interact with V1a, V2, and V1b receptors to exert its biological effects: V1a receptors have a strong effect of constricting small arteries; exciting V2 receptors can produce anti-diuretic effects, promote kidney water retention, and maintain Balance of osmotic pressure and cardiovascular homeostasis in vivo; V1b receptors are present in adenohypophysis and islet cells, and have endocrine mediating activity. AVP can regulate osmotic pressure, hemodynamics, blood coagulation, and affect endocrine function. AVP is a peptide hormone with neural effects. AVP secretion is affected by factors such as hyperosmolarity, hypovolemia, hypothalamic osmotic pressure receptors, and neuromodulation. It can be seen that AVP is involved in regulating the functions of the nervous system, cardiovascular system, endocrine system, and urinary system. The study further found that AVP levels in the blood of patients with cerebrovascular embolism, acute myocardial infarction, hypertension, and chronic obstructive pulmonary disease were significantly increased. Therefore, the determination of AVP in blood can be used to determine the occurrence of various diseases.

In blood circulation, AVP has a short half-life of 5-20 minutes; AVP in serum has poor in vitro stability and high storage requirements, making it difficult to accurately measure AVP. At present, radioimmunoassay is commonly used to measure serum AVP, because more than 90% of AVP is bound to platelets and is quickly cleared, which directly leads to a low detection result of AVP. The determination of AVP in serum by radioimmunoassay does not include AVP that binds to platelets, so the radioimmunoassay has low precision and large differences; in addition, AVP is a short peptide composed of 9 amino acids, which is not suitable for the determination of highly sensitive immunoassays, so it is necessary to find Substitute for AVP in blood. Proarginine vasopressin not only shears AVP, but also shears equimolar amount of CPP at the same time, and CPP and AVP are released into the blood in equimolar amount. The change trend of CPP and AVP is consistent, which reflects the release of AVP. Ideal replacement for levels.

CPP is a C-terminal part of proarginine vasopressin (126-164 amino acid sequence), consisting of 39 amino acids, a leucine-rich glycopeptide. CPP is produced in the paraventricular nucleus of the hypothalamus and released in pulses into the blood. Studies have shown that CPP plays an important role in AVP precursor processing, AVP maturation, transport and stabilization of AVP. CPP remains stable in the blood for a long time, has no enzyme cleavage site and receptor, is hardly degraded in the body, and is mainly excreted in the kidneys. CPP is more stable than AVP, and it is easier to measure by immunological methods. Therefore, the detection of CPP in blood is a surrogate index for AVP.

The study found that in the early stage of acute cerebrovascular infarction, the body is in a state of stress, which activates the hypothalamic-pituitary-adrenal axis (Hypothalamic-Pituitary-Adrenal Axis, HPA) "stress axis" system, in which the paraventricular nucleus of the hypothalamus is the activity of the HPA axis. At the direct control site, when stimulated by stress, parvocellular neurons in the paraventricular nucleus of the thalamus synthesize and secrete corticotropin-releasing hormone (CRH) and AVP. CRH and AVP reach the pituitary through the pituitary portal blood flow, and stimulate the pituitary to secrete adrenocorticotropic hormone (ACTH); ACTH reaches the adrenal gland through the blood circulation, stimulates the adrenal cortex to synthesize and secrete cortisol, and participates in the body's stress response.

Studies have found that bacterial, viral or fungal infections induce a host inflammatory response and induce high levels of IP-10 in the body's cells. The test found that the level of IP-10 in the serum of the normal group was less than 0.2ng/ml, the critical value of IP-10 in the serum of the bacterial infection group was 0.6ng/ml, and the critical value of IP-10 in the serum of the virus infection group was 1.9ng/ml. ml. Another study found that rhinovirus, respiratory syncytial virus, hepatitis B and C virus, and influenza virus infection induce high levels of IP-10 in body cells. In addition, serum IP-10 levels can be used to guide hepatitis C patients. A marker of treatment prognosis, therefore, detection of IP-10 levels in blood becomes a biomarker for the diagnosis of viral infection.

Therefore, in the prior art, a copeptin detection reagent based on immune layer analysis continues to be compatible with two specific binding reactions in time.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the shortcomings in the prior art, and propose a copeptin detection reagent based on immune layer analysis and a preparation method thereof.

To achieve the above object, the present invention provides the following technical solutions:

A copeptin detection reagent based on immunolayer analysis, including polystyrene nano-fluorescent microspheres, a solution of colored polystyrene microspheres, a coupling solution of anti- and peptidin antibodies, and a chromatographic solution. The content is 0.01%-1% Tween-20, the mass concentration is 0.1%-1.5% BSA and 1.5%-2.2% sucrose, and the solvent is phosphoric acid with a molar concentration of 0.015mol/l-0.035mol/l and a pH of 7.0-7.4 salt buffer.

Wherein, the carrier of the Copeptin detection reagent includes a detection test paper, and the detection test paper includes a PVC bottom plate and a sample pad pasted on the upper surface of the PVC bottom plate, an anti-Copeptin antibody tracer marker pad, a capture film, and a water-absorbing pad, Each adjacent section overlaps the edges at the edge joints.

Wherein, the sample pad is generally glass fiber, filter paper or blood filter membrane; the marker pad can be glass fiber, filter paper or polyester membrane; when the material of the sample pad and the marker pad are the same, they can be integrated together, using a glass fiber , filter paper or polyester membrane to complete the function of sample pad and marker pad.

Wherein, the capture membrane can be a nitrocellulose membrane (NC membrane), a nylon membrane, etc., the T line of the capture membrane contains small molecule organic compound antigens, the C line is a positive control, and the C line may contain a small molecule organic compound capable of detecting The secondary antibody of the antibody can also contain other proteins or antibodies that can be used as positive controls, such as biotin-labeled bovine serum albumin or ovalbumin (streptavidin on the label pad), or goat anti-rabbit antibody (label pads with rabbit antibodies).

A preparation method of copeptin detection reagent based on immunolayer analysis, comprising the following steps:

S1. Dilute carboxyl polystyrene nano-fluorescent microspheres and colored polystyrene microspheres with labeling buffer to a solid content of about 0.2%;

S2. Add EDC solution and Sulfo-NHS-LC-Biontin biotin labeling test solvent to the above-mentioned polystyrene nano-fluorescent microspheres and colored polystyrene microsphere solutions, activate for 15-30 minutes, centrifuge at high speed for 20 minutes, and remove the supernatant. Add an appropriate amount of anti-copeptin antibody solution, and mix and react for 1-1.5h;

S3, respectively use 0.1M, pH 7.0 phosphate buffer solution, and pH 9.0, 0.1M sodium borohydride solution as the coating solution to investigate the detection sensitivity and the background level after coating, and the detection sensitivity and background water select pH 7.0 phosphate The buffer solution is used as the coating solution of the FGF23 kit;

S4. Polystyrene nano-fluorescent microspheres, colored polystyrene microspheres solution and anti-copeptin antibody coupling solution, add blocking solution to react for 30-60 minutes, centrifuge at high speed for 20 minutes, remove supernatant, and add antibody preservation solution to obtain Anti-copeptin antibody polystyrene nano-fluorescent microspheres, colored polystyrene microspheres labeling solution;

S5, spray the above-obtained labeling solution onto glass fiber or polyester fiber with a gold-spraying machine, and the spray amount is 1-5 μL/cm. After drying and cutting, anti-copeptin antibody polystyrene nano-fluorescent microspheres are obtained. , Colored polystyrene microsphere labeling and peptide detection reagent.

Wherein, the preparation steps of the anti-copeptin antibody tracer labeling pad are: under the condition of magnetic stirring, adjust the pH value of 0.05 MK2CO3 to 8.0-8.5, add the anti-copeptin antibody dropwise, and slow down the rotation speed , and mix thoroughly for 30 min.

Among them, according to the final concentration of BSA mass ratio of 1% to 2%, add 10% BSA solution for blocking reaction, fully mix for 30 minutes; freeze and centrifuge at high speed for 30 minutes, discard the supernatant, and press 40% of the volume of the colloidal gold solution used in the original initial labeling. %～100% is added to the antibody preservation solution to reconstitute to obtain the anti-copeptin antibody labeling solution.

The anti-copeptin antibody labeling solution obtained above is sprayed on glass fiber or polyester fiber at a spray volume of 1-5 μL/cm, and the anti-copeptin antibody tracer labeling pad is obtained after drying and cutting.

Wherein, the preparation method of the detection test paper includes: taking out the adhesive backing, firstly adhering the treated NC film to the middle part of the adhesive backing, and secondly adhering the glass fiber paper treated with the marker as a bonding pad by lap bonding on the adhesive backing. On the top of the NC film, the untreated glass fiber paper is used as a sample pad to be lapped and bonded to the top of the bonding pad. Finally, the absorbent paper is lapped and bonded to the top of the NC film, and the test paper is cut with a slitter to a length of 6cm and a width of 4mm. of paper.

Wherein, the NC film contains a small molecule organic compound antigen, the small molecule organic compound is a steroid hormone and a fungal toxin, and the fungal toxin is one or both of aflatoxin or (and) zearalenone.

Technical effects and advantages of the present invention:

The invention provides a Copeptin detection reagent based on immune layer analysis and a preparation method thereof. By using the immune chromatography technology, the specific binding reaction of two antibodies forms a specific "antibody-antigen-antibody" immune complex, and then Combined with fluorescent or colored tracer labels, the reagent card can be used for quantitative or qualitative detection of capeptin in human blood samples when combined with a fluorometer;

The energy transition generated when the excited state returns to the ground state is direct chemiluminescence, without the participation of enzymes, rapid diagnosis, strong specificity, high sensitivity, easy storage, no need for special operators, and only simple instruments and equipment. The results. This will help medical staff to seize the best treatment time and play an active role in the diagnosis, condition and prognosis evaluation of stroke and heart failure patients.

Description of drawings

Fig. 1 is a flow chart of the preparation method of the copeptin detection reagent based on the immunolayer analysis of the present invention.

Detailed ways

In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Accordingly, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In the description of the present invention, it should be understood that the terms indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying what is indicated. A device or element must have a particular orientation, be constructed and operate in a particular orientation, and therefore should not be construed as limiting the invention.

In the present invention, unless otherwise expressly specified and limited, the terms "installed", "connected", "connected", "fixed" and other terms should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection , or integrated; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the internal connection of the two elements or the interaction relationship between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to the specific conditions of the description.

The present invention provides a copeptin detection reagent based on immune layer analysis, comprising polystyrene nano-fluorescent microspheres, a solution of colored polystyrene microspheres, a coupling solution of anti- and peptidin antibodies, and a chromatographic solution. The volume percentage of the elution liquid is 0.01%-1% Tween-20, the mass concentration is 0.1%-1.5% BSA and 1.5%-2.2% sucrose, and the solvent is a molar concentration of 0.015mol/l-0.035mol/l and a pH of 7.0 -7.4 Phosphate buffer.

Specifically, the carrier of the Copeptin detection reagent includes a detection test paper, and the detection test paper includes a PVC bottom plate and a sample pad pasted on the upper surface of the PVC bottom plate, an anti-Copeptin antibody tracer marker pad, a capture film, and a water-absorbing pad , and each adjacent part is overlapped at the edge joint.

Specifically, the sample pad is generally glass fiber, filter paper or blood filter membrane; the marker pad can be glass fiber, filter paper or polyester membrane; when the sample pad and the marker pad are of the same material, they can be integrated together, and a strip of glass Fiber, filter paper or polyester membrane to perform the function of sample pad and marker pad.

Specifically, the capture membrane can be a nitrocellulose membrane (NC membrane), a nylon membrane, etc., the T line of the capture membrane contains antigens of small molecular organic compounds, the C line is a positive control, and the C line may contain organic compounds that can bind to small molecules The secondary antibody for the detection antibody may also contain other proteins or antibodies that can be used as positive controls, such as biotin-labeled bovine serum albumin or ovalbumin (streptavidin on the label pad), or goat anti-rabbit antibody (labeled with rabbit antibodies on the mat).

Referring to FIG. 1, the present application also provides a preparation method of a Copeptin detection reagent based on immunolayer analysis, comprising the following steps:

S1. Dilute carboxyl polystyrene nano-fluorescent microspheres and colored polystyrene microspheres with labeling buffer to a solid content of about 0.2%;

S2. Add EDC solution and Sulfo-NHS-LC-Biontin biotin labeling test solvent to the above-mentioned polystyrene nano-fluorescent microspheres and colored polystyrene microsphere solutions, activate for 15-30 minutes, centrifuge at high speed for 20 minutes, and remove the supernatant. Add an appropriate amount of anti-copeptin antibody solution, and mix and react for 1-1.5h;

S3, respectively use 0.1M, pH 7.0 phosphate buffer solution, and pH 9.0, 0.1M sodium borohydride solution as the coating solution to investigate the detection sensitivity and the background level after coating, and the detection sensitivity and background water select pH 7.0 phosphate The buffer solution is used as the coating solution of the FGF23 kit;

S4. Polystyrene nano-fluorescent microspheres, colored polystyrene microspheres solution and anti-copeptin antibody coupling solution, add blocking solution to react for 30-60 minutes, centrifuge at high speed for 20 minutes, remove supernatant, and add antibody preservation solution to obtain Anti-copeptin antibody polystyrene nano-fluorescent microspheres, colored polystyrene microspheres labeling solution;

S5, spray the above-obtained labeling solution onto glass fiber or polyester fiber with a gold-spraying machine, and the spray amount is 1-5 μL/cm. After drying and cutting, anti-copeptin antibody polystyrene nano-fluorescent microspheres are obtained. , Colored polystyrene microsphere labeling and peptide detection reagent.

Specifically, the preparation steps of the anti-copeptin antibody tracer labeling pad are: under the condition of magnetic stirring, adjust the pH value of 0.05 MK2CO3 to 8.0-8.5, add anti-copeptin antibody dropwise, slow down speed and mix thoroughly for 30 min.

Specifically, according to the final BSA concentration mass ratio of 1% to 2%, add 10% BSA solution for blocking reaction, and mix thoroughly for 30 minutes; freeze and centrifuge at high speed for 30 minutes, discard the supernatant, and press the volume of the colloidal gold solution used in the original initial labeling. Add 40% to 100% of the antibody preservation solution to reconstitute to obtain an anti-copeptin antibody labeling solution.

Specifically, the anti-copeptin antibody labeling solution obtained above was sprayed onto glass fiber or polyester fiber with a gold-spraying stripper at a spray volume of 1-5 μL/cm, and the anti-copeptin antibody was obtained after drying and cutting. Tracer marker pad.

Specifically, the preparation method of the detection test paper includes: taking out the adhesive backing, firstly adhering the treated NC film to the middle part of the adhesive backing, and then lap bonding the glass fiber paper treated with the marker as a bonding pad On the top of the NC film, the untreated glass fiber paper was used as a sample pad to lap and stick on the top of the bonding pad, and finally the absorbent paper was lap-bonded on the top of the NC film, and the test paper was cut with a slitter to a length of 6 cm and a width of 6 cm. 4mm strips of paper.

Specifically, the NC film contains a small molecule organic compound antigen, the small molecule organic compound is a steroid hormone and a mycotoxin, and the mycotoxin is one or both of aflatoxin or (and) zearalenone .

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

It should be noted that, herein, the terms "comprising", "comprising" or any other variation thereof are intended to encompass non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements, It also includes other elements not expressly listed or inherent to such a process, method, article or apparatus.

Claims (10)

1. a copeptin detection reagent based on immune layer analysis, is characterized in that, comprises polystyrene nano-fluorescent microspheres, colored polystyrene microsphere solution and anti-, peptidin antibody coupling solution and chromatographic fluid, so The volume percentage of the chromatographic solution is 0.01%-1% Tween-20, the mass concentration is 0.1%-1.5% BSA and 1.5%-2.2% sucrose, and the solvent is a molar concentration of 0.015mol/l-0.035mol/l, pH Phosphate buffer for 7.0-7.4. 2. A kind of Copeptin detection reagent based on immune layer analysis according to claim 1, it is characterized in that: the carrier of described Copeptin detection reagent comprises detection test paper, and described detection test paper comprises PVC bottom plate and is pasted on PVC The sample pad on the upper surface of the bottom plate, the anti-copeptin antibody tracer label pad, the capture membrane, and the water-absorbing pad, each adjacent part is overlapped and bordered at the border. 3. The immunolayer analysis-based copeptin detection reagent according to claim 1, wherein the sample pad is generally glass fiber, filter paper or a blood filter membrane; the marker pad can be glass fiber, filter paper Or polyester film; when the sample pad and marker pad are of the same material, they can be integrated together, and a piece of glass fiber, filter paper or polyester film can be used to complete the function of the sample pad and the marker pad. 4. a kind of Copeptin detection reagent based on immunolayer analysis according to claim 1, is characterized in that: described capture membrane can be nitrocellulose membrane (NC membrane), nylon membrane etc., and described capture membrane The T line contains small molecule organic compound antigens, and the C line is a positive control. The C line may contain a secondary antibody that can bind to the detection antibody of small molecule organic compounds, or may contain other proteins or antibodies that can be used as positive controls, such as biotin-labeled bovine Serum albumin or ovalbumin (streptavidin on marker pad), or goat anti-rabbit antibody (rabbit antibody on marker pad). 5. A method for preparing a Copeptin detection reagent based on immune layer analysis according to any one of claims 1-4, characterized in that, comprising the steps of: S1. Dilute carboxyl polystyrene nano-fluorescent microspheres and colored polystyrene microspheres with labeling buffer to a solid content of about 0.2%; S2. Add EDC solution and Sulfo-NHS-LC-Biontin biotin labeling test solvent to the above-mentioned polystyrene nano-fluorescent microspheres and colored polystyrene microsphere solutions, activate for 15-30 minutes, centrifuge at high speed for 20 minutes, and remove the supernatant. Add an appropriate amount of anti-copeptin antibody solution, and mix and react for 1-1.5h; S3, respectively use 0.1M, pH 7.0 phosphate buffer solution, and pH 9.0, 0.1M sodium borohydride solution as the coating solution to investigate the detection sensitivity and the background level after coating, and the detection sensitivity and background water select pH 7.0 phosphate The buffer solution is used as the coating solution of the FGF23 kit; S4. Polystyrene nano-fluorescent microspheres, colored polystyrene microspheres solution and anti-copeptin antibody coupling solution, add blocking solution to react for 30-60 minutes, centrifuge at high speed for 20 minutes, remove supernatant, and add antibody preservation solution to obtain Anti-copeptin antibody polystyrene nano-fluorescent microspheres, colored polystyrene microspheres labeling solution; S5, spray the above-obtained labeling solution onto glass fiber or polyester fiber with a gold-spraying machine, and the spray amount is 1-5 μL/cm. After drying and cutting, anti-copeptin antibody polystyrene nano-fluorescent microspheres are obtained. , Colored polystyrene microsphere labeling and peptide detection reagent. 6 . The method for preparing a Copeptin detection reagent based on immune layer analysis according to claim 5 , wherein the preparation step of the anti-Copeptin antibody tracer labeling pad is: under magnetic stirring conditions. 7 . Then, adjust the pH value of 0.05 MK2CO3 to 8.0-8.5, add anti-copeptin antibody dropwise, slow down the rotation speed, and mix well for 30min. 7 . The method for preparing a Copeptin detection reagent based on immunolayer analysis according to claim 5 , wherein the final concentration of BSA is 1% to 2% by mass, and 10% BSA solution is added to carry out the blocking reaction. 8 . , fully mixed for 30min; refrigerated high-speed centrifugation for 30min, discarded the supernatant, added 40% to 100% of the volume of the colloidal gold solution used in the original initial labeling to the antibody preservation solution to reconstitute to obtain the anti-copeptin antibody labeling solution. 8. the preparation method of a kind of Copeptin detection reagent based on immune layer analysis according to claim 7, is characterized in that: the anti-copeptin antibody labeling solution obtained above is sprayed onto glass fiber with a gold-spraying stripper Or polyester fiber, the spray volume is 1-5 μL/cm, and the anti-copeptin antibody tracer marker pad is obtained after drying and cutting. 9 . The method for preparing a Copeptin detection reagent based on immune layer analysis according to claim 5 , wherein the method for preparing the detection test paper comprises: taking out a viscous substrate, and maximizing the processed NC film. 10 . First stick to the middle part of the adhesive backing, secondly, use the glass fiber paper treated with the marker as a bonding pad to lap and stick to the top of the NC film, and use the untreated glass fiber paper as a sample pad to lap and stick to the top of the bonding pad. On the top, the absorbent paper was finally overlapped and adhered to the top of the NC film, and the test paper was cut with a slitter to a length of 6cm and a width of 4mm. 10 . The method for preparing a Copeptin detection reagent based on immune layer analysis according to claim 9 , wherein the NC membrane contains a small molecule organic compound antigen, and the small molecule organic compound is a steroid hormone and a steroid hormone. 11 . Mycotoxins, which are one or both of aflatoxins or (and) zearalenone.

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