KR20150138968A - Plate for diagnosis and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a plate for diagnosis and a manufacturing method thereof and, more specifically, to a plate for diagnosis in which character capable of specifically binding to a target substance of an antibody is maximally preserved because the antibody can be directionally bound, and thus stability, uniqueness and sensitivity are improved, and to a manufacturing method thereof.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a diagnostic plate,

More particularly, the present invention relates to a diagnostic plate and a method for producing the same, and more particularly, to a method for producing a diagnostic plate and a method for producing the same, To a diagnostic plate having improved sensitivity and a method of manufacturing the same.

Antibodies have been used extensively in medical research and in the analysis of biological materials related to the diagnosis and treatment of diseases because they bind very specifically to antigens. In recent years, as an immunological measurement method, an immune sensor has been developed which immobilizes an antibody on a solid support material and measures changes in current, resistance, mass, and optical characteristics. Among them, immune sensors based on surface plasmon resonance using optical features have been commercialized. Biosensors based on surface plasmon resonance have been used for both qualitative information (whether two molecules specifically bind) and quantitative information Kinetics and equilibrium constants as well as detectable in real time without a label and are particularly useful for measuring antigen and antibody binding.

Among the immunosensors based on the surface plasmon resonance, an invention relating to a protein quantification method using Surface Plasmon Resonance (SPR), that is, Describes a method of quantifying the target protein by an SPR sensor and a method of increasing the sensitivity of the SPR signal by using a second amplification method using gold nanoparticles and an enzyme precipitation reaction.

On the other hand, in immunosensors, it is very important to selectively and stably immobilize an antibody on a solid support material. There are two major techniques for immobilizing antibodies, a chemical immobilization method and a physical immobilization method. Physical methods (Trends. Anal. Chem. 2000, 19, 530-540) are rarely used because they are low in reproducibility and denature proteins, and chemical methods (Langumur, 1997, 13, 6485-6490) And the reproducibility is good and the application range is widely used. However, when the antibody is immobilized by a chemical method, since the antibody is an asymmetric macromolecule, there arises a problem that the antibody loses its directionality or loses its binding activity with the antigen.

In order to improve the antigen binding ability of the antibody, which is the above-mentioned problem, there is a case where a support is used before binding the antibody to a solid support material, and a technique using protein G as such a support is known. However, when such a protein G itself is also bound to a support, there is a problem that its ability to bind to the antibody decreases because it is lost its directionality.

Therefore, various methods have been proposed to solve such problems. For example, staphylococcal protein G is treated with 2-iminothiolane to phosphorylate the amino acid group of the protein, followed by fixing the antibody using phosphorylated staphylococcal protein G. However, this method does not phosphorylate a specific part but phosphorylates the amino group of an amino acid having an amino group (Arg, Asn, Gln, Lys), so that the specificity is lowered and the purification process is required after chemical treatment.

On the other hand, a method of using DNA to immobilize a protein to be immobilized has been widely used. The DNA surface is known to be more stable and easier to manufacture than a protein chip. When the protein is immobilized using DNA, it is difficult to store it for a long period of time, immobilize the unstable protein, or store the protein under unstable conditions.

In addition, other methods of immobilizing antibodies on the surface of DNA have been reported. The first is immobilization of an antibody with a biotin attached thereto using a stripitavidin-DNA conjugate. Further, the antibody can be immobilized on the surface of the gold thin film to which the DNA is bound using a DNA linker. Both methods, however, have the disadvantage of losing the directionality of the antibody or possibly chemically altering the site to which the antigen is bound, since small molecules or DNA must be bound to the antibody.

The object of the present invention is to solve the problem of losing directionality when the antibody binds in the immunosensor as described above. The object of the present invention is to provide an antibody-specific binding protein capable of binding antibodies in a direction, To thereby maximize preservation of properties that can be specifically bound, thereby improving stability, specificity and sensitivity, and a method for producing the same.

Forming a parylene-H layer on one side of the substrate; Introducing an antibody-specific binding protein into the other surface of the parylene-H layer, and laminating the antibody-specific binding protein layer; Binding antibody to the antibody-specific binding protein layer to form a directional antibody layer to prepare a diagnostic plate; And binding a protein to the diagnostic plate to prevent a nonspecific reaction; The present invention also provides a method of manufacturing a diagnostic plate.

According to a preferred embodiment of the present invention, the forming of the parylene-H layer comprises: vaporizing the parylene-H dimer at 100 to 150 ° C for 30 to 60 minutes; Pyrolyzing the vaporized parylene-H dimer to para-xylene monomer at 500 to 700 ° C for 60 to 150 minutes; And depositing the para-xylene monomer on one side of the substrate at 15 to 25 ° C and 0.0001 to 0.01 torr to form a parylene-H layer; . ≪ / RTI >

According to another preferred embodiment of the present invention, the step of laminating the antibody-specific binding protein layer may be carried out by introducing 100-200 μl of antibody-specific protein into the other side of the parylene-H layer.

According to another preferred embodiment of the present invention, in the step of preparing the diagnostic plate, a directional antibody layer may be formed by introducing 100-200 μl of the antibody solution containing the antibody into the antibody-specific binding protein layer.

The present invention also relates to a parylene-H layer; A substrate laminated on one surface of the parylene-H layer; An antibody-specific binding protein layer laminated on the other side of the parylene-H layer; And an antibody conjugated with the antibody nonspecific binding protein layer; Wherein the substrate contains a protein that prevents a nonspecific reaction, the antibody-specific binding protein specifically binds to the Fc segment of the antibody, and comprises a directional antibody.

According to a preferred embodiment of the present invention, the antibody-specific binding protein comprises at least one member selected from the group consisting of protein G (protain G), protein A / G (protein A / G) and protein L can do.

According to another preferred embodiment of the present invention, the protein that prevents the nonspecific reaction is selected from the group consisting of bovine serum albumin, human serum albumin, and non-fat dry milk powder. And at least one of the groups.

According to another preferred embodiment of the present invention, the parylene-H layer may have an average thickness of 10 to 30 nm.

Further, the present invention provides a method for analyzing an antigen using the diagnostic plate as described above.

In addition, the present invention provides a biosensor including the above-described diagnostic plate.

The present invention relates to a diagnostic plate containing an antibody-specific binding protein capable of directionally binding an antibody to maximize preservation of properties capable of specifically binding to a target substance of an antibody, thereby improving stability, specificity, And a method for producing the same.

Fig. 1 shows the result of performing an antigen detection experiment using the diagnostic plate of Example 1 in Experimental Example 1. Fig.
FIG. 2 shows the result of performing an antigen detection experiment using the diagnostic plate of Example 2 in Experimental Example 1. FIG.
FIG. 3 shows the result of performing an antigen detection experiment using the diagnostic plate of Example 1 or Example 2 and the antigen solution of various concentrations in Experimental Example 2. FIG.

Hereinafter, the present invention will be described in more detail.

As described above, the conventional method of binding an antibody by converting a protein amino acid group into a phosphorylated group has a problem that the antibody-specific binding of the protein is reduced due to the phosphorylation of the amino acid group, and the method using DNA is stored for a long time , The immobilization of unstable proteins, and the storage of proteins under unstable conditions. Further, the method of binding small molecules or DNA to the antibody has a problem that the directionality of the antibody is lost or the part to which the antigen is bound can be chemically modified.

Accordingly, the present invention provides a method of manufacturing a semiconductor device, comprising: forming a parylene-H layer on one surface of a substrate; Introducing an antibody-specific binding protein into the other surface of the parylene-H layer, and laminating the antibody-specific binding protein layer; Binding antibody to the antibody-specific binding protein layer to form a directional antibody layer to prepare a diagnostic plate; And binding a protein to the diagnostic plate to prevent a nonspecific reaction; The present invention has been made in view of the above problems occurring in the prior art. A diagnostic plate including an antibody-specific binding protein capable of binding antibodies in a directional manner to maximize preservation of properties that can bind specifically to a target substance of the antibody, thereby improving stability, specificity, and sensitivity; and There is an effect of providing a manufacturing method thereof.

First, a parylene-H layer is formed on one surface of a substrate.

The substrate is not particularly limited as long as it is usually used as a diagnostic plate. For example, metals such as iron, steel, aluminum, copper, zinc, tin, lead, nickel, tin, gold or silver, glass, silicon, paper and polymers may be used.

Generally, parylene is a polymer in which p-xylene is polymerized, and has transparency. Parylene which is commonly used is generally referred to as parylene. Examples of derivatives of parylene N include parylene-A in which an amine group is linked to p-xylene, parylene-F in which fluorine is added, and parylene-H having a formyl group. It is particularly preferred that R is parylene-H.

In one embodiment of the present invention, the forming of the parylene-H layer comprises: vaporizing a parylene-H dimer at 100 to 150 ° C for 30 to 60 minutes; Pyrolyzing the vaporized parylene-H dimer to para-xylene monomer at 500 to 700 ° C for 60 to 150 minutes; And depositing the para-xylene monomer on one side of the substrate at 15 to 25 ° C and 0.0001 to 0.01 torr to form a parylene-H layer.

First, the parylene-H dimer is vaporized.

The vaporization is not particularly limited as long as it is a condition commonly used in the parylene-H dimerization, but it is preferably carried out at 100 to 150 ° C for 30 to 60 minutes.

If the temperature is lower than 100 ° C., the parylene-H dimer may not vaporize. If the temperature exceeds 150 ° C., the parylene-H dimer is vaporized at a high rate, A problem that a film is formed may occur.

If the vaporization is performed for less than 30 minutes, there is a problem that the thickness of the deposited parylene-H dimer is vaporized to be smaller than the amount of the deposited film, and when vaporized for more than 60 minutes, There may arise a problem that the Rhen-H dimer is vaporized and forms a thicker film than the film to be deposited.

Next, the vaporized parylene-H dimer is pyrolyzed with a para-xylene monomer.

The pyrolysis is not particularly limited as long as it is a condition for pyrolyzing the parylene-H dimer. The pyrolysis can be performed preferably at 500 to 700 ° C for 60 to 150 minutes.

If the temperature is lower than 500 ° C, the parylene-H dimer may not be completely pyrolyzed with the monomer to form a film in a dimer state. If the temperature exceeds 700 ° C, the parylene- There is a problem that the film is damaged due to heat and a non-uniform film is deposited.

If pyrolysis is carried out for less than 60 minutes, the vaporized parylene-H dimer may be partially pyrolyzed with monomers to form a thinner film than the film to be deposited. If pyrolysis occurs for more than 150 minutes, A larger amount of the parylene-H dimer may be pyrolyzed with the monomer to form a thicker film than the film to be deposited.

Next, the para-xylene monomer is deposited on one side of the substrate to form a parylene-H layer.

The deposition is not particularly limited as long as it is a condition commonly used for vapor deposition of radicals or gaseous monomers of a gas, but it is preferably carried out at 15 to 25 ° C and 0.0001 to 0.01 torr.

If the temperature is lower than 15 ° C., the deposition rate is greatly increased. Therefore, it may become difficult to control the deposition time to obtain a film having a desired thickness. When the temperature exceeds 25 ° C., So that it takes much time to deposit the film.

If the deposition is performed at a pressure lower than 0.0001 torr, it may take a long time to lower the pressure depending on the performance of the vacuum pump. When the deposition is performed at a pressure exceeding 0.01 torr, the parylene-H film becomes opaque and non- A problem of deposition may occur.

The thickness of the parylene-H layer is not particularly limited, but may be preferably 5 to 50 nm, and more preferably 10 to 30 nm.

If the average thickness is less than 5 nm, there is a problem that the target surface is not partially deposited. If the average thickness exceeds 50 nm, the thickness exceeds the thickness at which the surface plasmon resonance can be measured So that it is impossible to perform a measurement experiment using a surface plasmon resonance sensor.

It will be understood by those skilled in the art that the parylene-H thin film can be applied to the plate substrate by various methods other than the above method.

Next, an antibody-specific binding protein is introduced into the other side of the parylene-H layer to laminate the antibody-specific binding protein layer.

The antibody-specific binding protein serves to bind the antibody directionally, and is not particularly limited as long as it is a protein having a specific binding characteristic to the Fc segment of an antibody. Preferably, the antibody-specific binding protein is protein G (protain G) And may include at least one of proteins A / G (protein A / G) and protein L (protein L), and more preferably protein G (protain G).

The lamination is not particularly limited as long as it is a condition to be used for binding a polymer and a protein, but it is preferably carried out by introducing 100-200 μl of an antibody-specific protein on the other side of the parylene-H layer.

If the antibody-specific protein of less than 100 μL is introduced, the ability to immobilize the antibody is decreased due to the partial lamination of the protein on the surface of the parylene-H layer due to the lack of the antibody-specific protein amount And when an antibody specific protein exceeding 200 μl is introduced, the problem may arise that the protein can not be stacked beyond the amount of the antibody-specific protein capable of saturating the surface of the parylene-H layer .

Next, an antibody is bound to the antibody-specific binding protein layer to form a directional antibody layer to prepare a diagnostic plate.

The antibody can be suitably selected according to the target antigen or the target detection substance, and the type of the antibody is not limited.

The directional antibody layer forming conditions are not particularly limited as long as the conditions used for binding the antibody and the protein are normally, but a directional antibody layer can be formed by introducing 100-200 μl of the antibody solution containing the antibody.

If an antibody solution of less than 100 μl / min is used, the antibody may be partially deposited on the surface of the antibody-specific protein layer due to the lack of the antibody, resulting in a reduced ability to detect a target substance for diagnosis When an antibody solution of more than 200 μl is used, a problem may occur that some antibodies can not be stacked and consumed in excess of the amount of antibody capable of saturating the antibody-specific protein layer.

The antibody solution is not particularly limited as long as it includes an antibody and a solvent capable of transporting the antibody to the protein. Preferably, the antibody solution may include antibodies at a concentration of 100 to 200 μg / ml.

If a solution having a concentration of less than 100 占 퐂 / ml is used, the antibody may partially be laminated on the surface of the antibody-specific protein layer due to the lack of the antibody, thereby reducing the ability to detect the target substance for diagnosis When a solution having a concentration of more than 200 μg / ml is used, a problem may occur that some antibodies can not be stacked and consumed in excess of the amount of antibody capable of saturating the antibody-specific protein layer.

The solvent is not particularly limited as long as it is usually used for antibody-protein binding. For example, phosphate buffered saline, Tris-HCl, TBE (Tris base, boric acid and ethylenediaminetetraacetic acid (EDTA) mixture) or Tris-Buffered Saline (TBS) can be used.

Next, a protein preventing nonspecific reaction is bound to the diagnostic plate.

The protein that prevents the nonspecific reaction plays a role of preventing the reaction between a site not bound to the antibody and another biomolecule other than the target antigen and is not particularly limited as long as it is known that the reactivity with biomolecules is low in general Preferably, it may include at least one of the group consisting of bovine serum albumin, human serum albumin and non-fat dry milk powder, and most preferably, May contain bovine serum albumin.

The binding conditions are not particularly limited as long as they are used for protein binding. Preferably, 100-200 μl of a solution containing 50-150 μg / ml of protein for preventing nonspecific reaction may be injected.

If a solution having a concentration of less than 50 占 퐂 / ml is used, there is a problem that the amount of protein is insufficient so that the surface is not saturated and a part of the surface is exposed and nonspecific reaction can not be prevented. , There is a problem that some proteins can not be stacked and consumed in excess of the amount of protein that can saturate the surface.

If a solution of less than 100 μl is used, there may be a problem that the amount of the protein is insufficient and the surface is not saturated and a part of the surface is exposed, so that a nonspecific reaction can not be prevented. , The amount of the protein that can saturate the surface is exceeded, and some proteins may not be stacked and consumed.

Further, the present invention relates to a parylene-H layer; A substrate laminated on one surface of the parylene-H layer; An antibody-specific binding protein layer laminated on the other side of the parylene-H layer; And an antibody conjugated with the antibody nonspecific binding protein layer; Wherein the substrate contains a protein that prevents a nonspecific reaction, the antibody-specific binding protein specifically binds to the Fc segment of the antibody, and comprises a directional antibody.

The thickness of the parylene-H layer is not particularly limited, but may preferably be an average thickness of 5 to 50 nm, and more preferably an average thickness of 10 to 30 nm.

If the average thickness is less than 5 nm, there is a problem that the target surface is not partially deposited. If the average thickness exceeds 50 nm, the thickness exceeds the thickness at which the surface plasmon resonance can be measured So that it is impossible to perform a measurement experiment using a surface plasmon resonance sensor.

The substrate is not particularly limited as long as it is usually used as a diagnostic plate. For example, metals such as iron, steel, aluminum, copper, zinc, tin, lead, nickel, tin, gold or silver, glass, silicon, paper and polymers may be used.

The antibody can be suitably selected according to the target antigen or the target detection substance, and the type of the antibody is not limited.

The protein that prevents the nonspecific reaction plays a role of preventing the reaction between a site not bound to the antibody and another biomolecule other than the target antigen and is not particularly limited as long as it is known that the reactivity with biomolecules is low in general Preferably, it may include at least one of the group consisting of bovine serum albumin, human serum albumin and non-fat dry milk powder, and most preferably, May contain bovine serum albumin.

The antibody-specific binding protein serves to bind the antibody directionally, and is not particularly limited as long as it is a protein having a specific binding characteristic to the Fc segment of an antibody. Preferably, the antibody-specific binding protein is protein G (protain G) And may include at least one of proteins A / G (protein A / G) and protein L (protein L), and more preferably protein G (protain G).

In addition, the present invention provides a biosensor including the above-described diagnostic plate.

In addition, the present invention provides a method for analyzing an antigen using the diagnostic plate as described above.

As shown in Experimental Examples 1 and 2, antigen analysis using the diagnostic plate of the present invention showed that the diagnostic plate of the present invention had high antigen sensitivity and was suitable as an antigen diagnostic plate.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the invention is not limited by these examples.

[ Example ]

Example  1. Manufacture of diagnostic plates

Gold chips (BK7, K-MAC, Korea) were washed with ultrasonic waves of 40 kHz and 200 W for 5 minutes using acetone, methanol, deionized water (DI water) Thereafter, the washed gold chips were put in a coater (NRPC-500, Nuri-Tech, Korea) and parylene-H dimer (parylene-H dimer) ; Seoul, Korea) was added in a coater, and parylene was vaporized at 100 ° C for 40 minutes and pyrolyzed at 670 ° C for 110 minutes to form a para-xylene monomer. The para-xylene monomers were then deposited on gold chips at 25 ° C and 0.01 torr to deposit a parylene-H layer having an average thickness of 21 nm.

The surface plasmon resonance (SPR) sensor system (SPR micro, K-MAC, Korea), peristaltic micro-pump (REGLO, ISMATEC, USA) and degasser (DGU- 12A, SHIMADZU, Japan) was used to laminate an antibody-specific protein layer.

Specifically, a gold chip in which a parylene-H layer was laminated was placed on a 2-channel surface plasmon resonance sensor system, phosphate buffered saline was injected into a reference channel, and protein G (Sigma, MO, USA) (Sigma, MO, USA) at a flow rate of 30 μl / min for 5 minutes in a sample flow channel containing phosphate buffered saline (PBS) at a concentration of 10 μg / Binding protein layer. Protein G, which was weakly deposited or unreacted, was then washed with phosphate buffered saline (PBS).

150 μl of a phosphate buffered saline solution containing 150 μg / ml of a human IgG antibody (anti-human IgG; Sigma, MO, USA) was injected through a sample channel into a gold chip having an antibody-specific binding protein layer stacked thereon, . Then, the plate was washed with phosphate buffer saline (PBS) to prepare a diagnostic plate.

150 μl of a phosphate buffered saline solution containing 100 μg / ml of bovine serum albumin (manufactured by SIGMA) was injected into the diagnostic plate through a sample channel to bind a diagnostic plate and a protein preventing nonspecific reaction.

Example  2.

A diagnostic plate was prepared in the same manner as in Example 1, except that the antibody and the parylene-H layer were combined without binding Protein G.

Experimental Example  1. Antigen detection experiment

Using a 2-channel surface plasmon resonance sensor system, 150 μl of human IgG (human IgG; Sigma, MO, USA) solution was injected into the sample flow channel of the diagnostic plate prepared in Example 1 or Example 2, The reaction was carried out. Then, the antigen that did not bind to or weakly bound to the antibody of the diagnostic plate was removed with phosphate buffered saline. Then, in order to confirm the antigen detection efficiency of the diagnostic plate, the IgG detection signal of the diagnostic plate was measured and shown in FIGS. 1 to 2.

The human IgG solution used was a solution containing human IgG at a concentration of 3 μg / ml in phosphate-buffered saline. Further, the detection signal was calculated by subtracting the signal of the phosphate buffered saline flowing into the reference channel from the measured signal.

Fig. 1 shows results of detection of antigens using the diagnostic plate of Example 1, and Fig. 2 shows results of detection of antigens using the diagnostic plate of Example 2. Fig.

As can be seen from Figs. 1 and 2, it was confirmed that the use of the diagnostic plate of Example 1 significantly increased antigen detection sensitivity compared to the case of using the diagnostic plate of Example 2, which does not contain protein G .

Thus, the diagnostic plate of the present invention has a high antigen sensitivity, which makes it suitable for use as an antigen diagnostic plate.

Experimental Example  2. Detection efficiency according to antigen concentration

Using a 2-channel surface plasmon resonance sensor system, phosphate buffered saline was sufficiently flowed through the reference channel into the diagnostic plates prepared in Examples 1 and 2, and then the concentration of 1 / / ml, 3 / / ㎖ or 5 / / Lt; RTI ID = 0.0 > 100 < / RTI > second into a sample flow channel and the signal from the diagnostic plate was measured. In addition, signal measurement was performed using the same concentration of human IgG solution three times.

The measured signal is shown in FIG. 3, and the error bar in FIG. 3 shows the standard deviation for three experiments using the same concentration of solution.

3, it was confirmed that the detection signal of the diagnostic plate of Example 1 was significantly increased as compared with the diagnostic plate of Example 2 containing no protein G, The diagnostic plate of the present invention has high antigen detection sensitivity.

As can be seen from the Examples and Experimental Examples, the diagnostic plate of the present invention was found to be highly suitable as an antigen diagnostic plate due to its high antigen sensitivity.

Claims (10)

Forming a parylene-H layer on one surface of the substrate;
Introducing an antibody-specific binding protein into the other surface of the parylene-H layer, and laminating the antibody-specific binding protein layer;
Binding antibody to the antibody-specific binding protein layer to form a directional antibody layer to prepare a diagnostic plate; And
Binding a protein to the diagnostic plate to prevent nonspecific reaction;
≪ / RTI > The method of claim 1, wherein forming the parylene-H layer comprises:
Vaporizing the parylene-H dimer at 100 to 150 占 폚 for 30 to 60 minutes;
Pyrolyzing the vaporized parylene-H dimer to para-xylene monomer at 500 to 700 ° C for 60 to 150 minutes; And
Forming a parylene-H layer by depositing the para-xylene monomer on one side of the substrate at 15 to 25 DEG C and 0.0001 to 0.01 torr;
Wherein the method comprises the steps of: [2] The method of claim 1, wherein the step of laminating the antibody-specific binding protein layer is carried out by introducing 100-200 [mu] l of antibody-specific protein into the other side of the parylene-H layer. The diagnostic plate manufacturing method according to claim 1, wherein the step of preparing the diagnostic plate comprises introducing 100 to 200 μl of the antibody solution containing the antibody into the antibody-specific binding protein layer to form a directional antibody layer . A parylene-H layer;
A substrate laminated on one surface of the parylene-H layer;
An antibody-specific binding protein layer laminated on the other side of the parylene-H layer; And
An antibody conjugated with the antibody nonspecific binding protein layer; Lt; / RTI >
Wherein the substrate contains a protein that prevents nonspecific reaction,
The antibody-specific binding protein specifically binds to the Fc segment of the antibody,
A diagnostic plate comprising a directional antibody. The antibody-specific binding protein according to claim 5, wherein the antibody-specific binding protein comprises at least one member selected from the group consisting of protein G (protein G), protein A / G (protein A / G) A diagnostic plate. 6. The method of claim 5, wherein the protein that prevents the nonspecific reaction is one or more of the group consisting of bovine serum albumin, human serum albumin, and non-fat dry milk powder Wherein the plate is made of a metal. 6. The diagnostic plate according to claim 5, wherein the parylene-H layer has an average thickness of 5 to 50 nm. A method for analyzing an antigen using the diagnostic plate according to any one of claims 5 to 8. A biosensor comprising the diagnostic plate according to any one of claims 5 to 8.

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