JP2000206115A - Immunoagglutination reaction reagent using antibody fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent of a high sensitivity and a good accuracy by processing a monochlonal antibody or polychlonal antibody with a reducing agent, fragmenting the antibody and letting the fragment carried by an insoluble carrier through physical adsorption or chemical bond. SOLUTION: Not only a monoclonal antibody, but a polychlonal antibody are processed with a reducing agent, for example, 2-mercaptoethanol or the like, thereby fragmenting immunoglobulin. The antibody fragment is carried by an insoluble carrier. The reagent is generated in this manner. The carrier used can be any of that can carry the antibody, flocculates when the carrier after holding the antibody generates an antigen-antibody reaction, and enables a labelling substance to measure an activity in any method, for example, fine particles of organic polymer substances, functional group-introduced inorganic particles, organism-originated particles or the like. The reagent improves a reactivity and a linearity both in a low concentration region and a high concentration region.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a reagent for immunologically measuring a component in a test solution utilizing an antigen-antibody reaction, and by fragmenting an antibody used in the reagent, a wide measurement range can be obtained. The present invention relates to a reagent having the same and a production method.

[0002]

2. Description of the Related Art As a method for measuring a specific component in a test solution, there are a number of immunological measurement reagents utilizing an antigen-antibody reaction. Improvements have been attempted. In producing an immunoassay reagent, an untreated antibody is generally used as a material when an antibody against a specific component to be measured is carried on an insoluble carrier. To avoid the effects of non-specific reactions, Fac
There are also methods using b-fragment (JP-B-63-41426, JP-B-63-63863) and methods using F (ab ') ₂ and Fab fragments.

[0003] However, Fab and F (ab ') ₂ fragments contain F
Since the c-part is missing, there is a problem that it is harder to carry on an insoluble carrier than an untreated antibody. Also,
Some animal species cannot be processed into Facb fragments, and some cannot withstand enzyme treatment depending on the class of antibody.

[0004]

SUMMARY OF THE INVENTION The present invention relates to an immunoagglutination reagent having a reactivity equal to or higher than the conventional one, that is, a highly sensitive and accurate immunoagglutination reagent, a method for preparing the same, and a method for measuring the same. The purpose is to provide.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention is an invention comprising fragmenting immunoglobulins including not only monoclonal antibodies but also polyclonal antibodies and supporting the fragments on an insoluble carrier. The present invention also provides an immunological method comprising physically or chemically using a monoclonal antibody or a polyclonal antibody fragmented by treating it with a reducing agent, and carrying the antibody on an insoluble carrier. This is an invention of a method for preparing a measurement reagent.

Further, the present invention relates to the use of a monoclonal or polyclonal antibody fragmented by treating it with a reducing agent, and using an immunological assay reagent physically or chemically carrying the antibody. An invention of an immunological measurement method characterized by the following. That is, the present inventors,
When carrying immunoglobulin on an insoluble carrier, merely carrying an antibody is difficult for some antibodies to exhibit reactivity. I have been working hard. As a result, in addition to the monoclonal antibody, the method of fragmenting the immunoglobulin by treating the polyclonal antibody with a reducing agent and supporting it on an insoluble carrier, as compared with the conventional method of supporting an antibody on an insoluble carrier, They have found that better reactivity can be maintained, and have completed the present invention.

In the present invention, the above-mentioned means makes it possible to produce an immunoassay reagent having a wide measurement range and a small amount of antibody used. The reason is presumed as follows. By reducing the antibody, the SS bond connecting the two H chains at the hinge portion and the Fc site is released, and two molecules having one antigen recognition site are converted from one antibody having two antigen recognition sites Of half antibodies are produced. It is considered that steric hindrance and the like are reduced by using two molecules, and the number of antigen recognition sites involved in an actual antigen-antibody reaction is increased. Also,
Fragmentation with this reducing agent can also be produced from Facb fragments.

[0008] The antibody used in the present invention comprises:
Without limiting the animal species, for example, derived from mice, rats, rabbits, goats, sheep, humans, etc., besides monoclonal antibodies or polyclonal antibodies,
It is also possible to use a modified antibody such as a chimeric antibody, and the type of the antibody is not particularly limited. Commercially available immunoglobulins may be used, or those obtained from animal serum or cell culture supernatant by a known method may be used.

As the carrier used in the present invention, a known carrier can be used without any particular limitation as long as the carrier can carry an antibody and aggregates when the carrier after carrying out an antigen-antibody reaction. The amount of the supported antibody in preparing the intended supported insoluble carrier is not particularly limited, and may be smaller than the theoretically supportable amount or may be a large excess.

Further, the carrier used in the present invention can carry an antibody, and when the carrier after carrying the antigen-antibody reaction can measure the activity of the labeled substance by any method, Known carriers can be used without particular limitation.

[0011] Carriers that can be suitably used for the immunoagglutination reaction include:
Any insoluble carrier can be used as long as it is an insoluble carrier used in a usual immunoassay. For example, polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, styrene- Fine particles of organic polymer substances such as latex such as styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylate copolymer, and polyvinyl acetate acrylate Or, silica, silica-alumina, inorganic oxides such as alumina or an inorganic oxide or the like subjected to a silane coupling treatment or the like to introduce functional groups into inorganic particles, human O-type blood cells, sheep erythrocytes, or other biological sources. Examples include, but are not limited to, particles. Among them, latex particles are artificial carriers, and are particularly preferably used in view of the fact that the surface of the carrier is easily subjected to a chemical treatment and the non-specific reaction hardly occurs depending on the purpose. Of these, it goes without saying that various modified latexes, magnetic latexes, and the like can be used as needed.

Although the particle size of the carrier is not particularly limited, the average particle size is 0.05 to 10 μm from the viewpoint of the easiness of aggregation after the antigen-antibody reaction and the easiness of discrimination of the aggregation.
It is preferable to use a carrier. The amount of the carrier may be appropriately determined depending on the type of the antibody or the antigen,
From the viewpoints of loading efficiency and operability, it is preferable to use the solution in a concentration of 0.001 to 15 wt% during measurement, and it is generally used in the form of a suspension.

The antibody carried on the carrier in the present invention may be any antibody that causes an antigen-antibody reaction with the antigen to be tested. Antibodies preferably used in the present invention include, for example, anti-α1-m antibody, anti-pepsinogen antibody, anti-prostate specific antigen (PSA) antibody, anti-β2-m antibody, anti-fibrinogen antibody, anti-fibrin degradation product (FDP) ) Antibodies, anti-albumin antibodies, anti-human IgG antibodies, anti-C-reactive protein (CR
P) antibodies, anti-ferritin antibodies, anti-α-fetoprotein (AFP) antibodies, anti-insulin antibodies and the like.

As a method of supporting the antibody on a carrier, known methods can be used without particular limitation. Basic loading methods include a physical adsorption method and a chemical bonding method, and the physical adsorption method is suitably used in terms of the simplicity of the loading operation.

The immunological assay reagent of the present invention is a reagent that utilizes a phenomenon associated with an immune reaction that occurs when an antibody is carried on a carrier and brought into contact with an antigen in a sample. Examples of the qualitative reagent using the latex agglutination method include a latex agglutination reagent and a microtiter reagent, and examples of the quantitative reagent include a latex quantitative reagent that optically measures the degree of aggregation.

The solution containing the immunoglobulin fragment is not particularly limited, but is preferably a solution that does not prevent the immunoglobulin fragment from adsorbing or binding to an insoluble carrier. For example, purified water, or pH
5.0 to 10.0, more preferably pH 6.5 to 8.5.
Phosphate buffer, Tris buffer, Good buffer, glycine buffer, borate buffer and the like having a buffering action near neutrality are exemplified. The concentration of these buffers is usually 10
The concentration is appropriately selected from the range of -500 mM, preferably 10-200 mM. Further, in this solution, for example, sugars, salts such as sodium chloride, surfactants, preservatives, proteins, and the like are contained as long as the amount does not prevent the immunoglobulin fragments from adsorbing or binding to the insoluble carrier. May be.

The reducing agent used in the present invention is not particularly specified, but may be any as long as it has a reducing action. For example, 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, dithiothreitol (DTT) and the like can be mentioned. The concentration may be any concentration that can reduce immunoglobulins to half molecules, and is appropriately selected depending on the properties of the antibody. Further, in order to make the reducing action more effective, a denaturant such as urea, sodium dodecyl sulfate (SDS), Triton X or the like may be used in the reaction solution, and the concentration thereof is appropriately selected depending on the properties of the antibody.

The immunoglobulin fragment used in the present invention can be determined by analyzing it by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis image shows a molecular weight of about 150,000 when immunoglobulin not treated with a reducing agent was used as a sample, but a fragmented sample had a molecular weight of about 70,000.
You can check before and after. In addition, when the immunoglobulin fragment is physically supported on an insoluble carrier and then isolated with a surfactant or the like, the immunoglobulin fragment is identified by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). it can.

Next, a reagent form of a latex quantitative reagent will be described as an example of the immunoagglutination reagent of the present invention. It does not do.

A mixed solution containing the following components (1), (2) and (3) as basic components: (1) Buffer solution: 20 to 1000 mM, pH 4 to 12 (2) Carrier supporting immunoglobulin fragment 0.01 to
0.5% (w / v) (3) Examples of the 50 to 300 mM sodium chloride buffer include Tris buffer, phosphate buffer, glycine buffer, borate buffer and Good buffer.

As an example of the method for producing the above reagent, the following method can be exemplified. First, 0.5 to 10% latex particles
(W / v) After mixing a buffer containing 0.05 to 100 mg / ml of the immunoglobulin fragment with the suspended buffer,
Shake for 30 minutes to 48 hours at 4 to 60 ° C. Thereafter, the carrier and the solution are separated by centrifugation or the like to remove unbound immunoglobulin fragments. By washing the obtained latex particles with the above buffer solution, the desired immunoglobulin fragment-carrying latex particles can be obtained. Here, a blocking treatment, that is, a treatment of immersing in a solution containing proteins not involved in the reaction, for example, bovine serum albumin (hereinafter abbreviated as BSA), milk protein, ovalbumin, and denatured products thereof, is performed. It is desirable to prevent non-specific reactions. Next, a dispersion medium for maintaining the dispersibility required for the measurement is added, and the carrier is dispersed in a buffer solution to obtain the above reagent.

Next, a method for producing the immunoglobulin fragment of the present invention will be described below. First, immunoglobulin 0.05 ~
The buffer containing 100 mg / ml is degassed in a deoxygenated state, for example, under reduced pressure. 30 minutes to 48 hours after adding a reducing agent, for example, 1 to 20 μl of 2-mercaptoethanol, which is appropriately selected according to the properties of the antibody, 4 to 60
C., preferably at 20 to 37.degree. Thereafter, a substance that blocks a free SH group, for example, N-ethylmaleimide or the like is appropriately added to prevent re-association of the SH group. Using a buffer containing EDTA,
The target immunoglobulin fragment is collected by using a column for molecular weight fractionation or the like.

[0023]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the scope described in these examples.

Example 1 (1) Anti-human prostate specific antigen antibody (anti-human PSA antibody)
Reduction process. The anti-human PSA monoclonal antibodies A, B, C, and D were each adjusted to a concentration of 10 mg / ml at a pH of 0.2.
Prepared in 01M phosphate buffer (hereinafter abbreviated as PBS),
1.5 μl of 2-mercaptoethanol (Wako Pure Chemical Industries) is added to 1 ml. After incubating at 37 ° C. for 90 minutes, 0.01 mg of N-ethylmaleimide (Sigma) is added. After incubation at 37 ° C. for 10 minutes, the desired fraction can be obtained by gel filtration using a Superdex 200 (Pharmacia) column with 20 mM EDTA and 0.1 M phosphate buffer. This immunoglobulin fragment was used for the following preparation.

(2) Preparation of a fragmented anti-PSA antibody-carrying latex suspension by reduction treatment. Polystyrene particles having an average particle diameter of 0.3 μm were diluted with PBS having a pH of 7.2 to prepare 2 ml of a suspension having a latex concentration of 5% (w / v). Then the above fragmented anti-PS
A solution obtained by diluting the monoclonal antibody of A with PBS (10
(mg / ml) was added and mixed. After shaking at 37 ° C. for 4 hours, unreacted antibodies were removed by centrifugation.
1 ml of an aqueous solution containing 5% of SA was added. Then 99m
1 M of a 0.1 M HEPES buffer solution containing 0.1 M sodium chloride and 0.1% sodium azide at pH 8.0 (hereinafter, this mixed solution is abbreviated as HEPES-S) was added to prepare a suspension.

(3) Preparation of PSA test solution. 0.1 M HEPES-S, PSA antigen solution was added to 0.1 M
And diluted with HEPES-S to a concentration of 0.0,0. 21,0.8
6,2.59,7.77,23.3,70,210ng
/ Ml and used as a test solution.

(4) Measurement method. After adding 20 μl of the test solution to 300 μl of the above suspension in a plastic cell and stirring, the wavelength 5 at 37 ° C. for about 10 minutes was added.
The amount of change in absorbance at 46 nm was measured. The automatic analyzer Hitachi 7070 was used for the above operations.

(5) Relationship with the change in absorbance. The PSA test solution was measured with the above-prepared reagent and the anti-PSA antibody-carrying latex suspension fragmented by the reduction treatment, and the results are shown in Table 1.

Comparative Example 1 (1) Preparation of anti-PSA antibody-carrying latex suspension. A polystyrene particle having an average particle diameter of 0.3 μm is diluted with PBS to give a suspension having a latex concentration of 5% (w / v) in 2 ml.
Prepared. Then, the anti-PSA monoclonal antibody was
1 ml of a solution (10 mg / ml) diluted with S was added and mixed. After shaking at 37 ° C. for 4 hours, unreacted antibodies were removed by centrifugation, and 1 ml of an aqueous solution containing 5% BSA was added. Then, 0.1 ml of pH 8.0 containing 99 ml of 0.1 M sodium chloride and 0.1% sodium azide.
Was prepared by adding and suspending HEPES-S.

(2) Preparation and measurement of PSA test solution. Preparation and measurement were performed in the same manner as for the fragmented PSA antibody-carrying latex. Table 1 shows the results.

[0031]

[Table 1]

Example 2 (1) Reduction treatment of anti-human albumin antibody (anti-human Alb antibody). Anti-human Alb monoclonal antibody A was adjusted to a concentration of 10 mg / ml in a 0.01 M phosphate buffer (pH 7.2).
And 1.5 μl of 2-mercaptoethanol (Wako Pure Chemical Industries) is added to 1 ml. 90 at 37 ° C
After incubating for N minutes, N-ethylmaleimide (Si
gma) is added at 0.01 mg. After incubating at 37 ° C. for 10 minutes, 20 mM EDTA, 0.1 M was prepared using a Superdex200 (Pharmacia) column.
The desired fraction can be obtained by gel filtration with a phosphate buffer. This immunoglobulin fragment was used for the following preparation.

(2) Reduction treatment of anti-human Alb antibody Facb fragment. Anti-human Alb monoclonal antibody A was prepared in PBS to a concentration of 10 mg / ml, and the pH was adjusted to 2.5 with hydrochloric acid.
To be prepared. After incubating at 30 ° C for 60 minutes,
The pH is returned to 7.0 with sodium hydroxide and
Incubate for minutes to obtain the Facb fragment. 1.5 μl per 1 ml of this fragment solution
Of 2-mercaptoethanol (Wako Pure Chemical Industries) is added. After incubating at 37 ° C. for 90 minutes, 0.01 mg of N-ethylmaleimide (Sigma) is added. 3
After incubation at 7 ° C for 10 minutes, Superdex
20 mM E using a 200 (Pharmacia) column
The desired fraction can be obtained by gel filtration with DTA and 0.1 M phosphate buffer. This immunoglobulin fragment was used for the following preparation.

(3) Preparation of a latex suspension carrying a fragmented anti-Alb antibody by reduction treatment. Polystyrene particles having an average particle size of 0.12 μm are diluted with PBS to give a suspension having a latex concentration of 5% (w / v) in 2 m.
1 was prepared. Next, a solution (10 mg / m2) of the anti-Alb monoclonal antibody fragmented in (1) diluted with PBS
l) was added and mixed. After shaking at 37 ° C. for 4 hours, unreacted antibodies were removed by centrifugation, and BSA 5%
1 ml of an aqueous solution containing Then 99 ml of 0.
It was prepared by adding and suspending 0.1 M HEPES (pH 8.0) containing 1 M sodium chloride and 0.1% sodium azide.

(4) Preparation of a latex suspension carrying a fragmented anti-Alb antibody by reducing the Facb fragment. Polystyrene particles having an average particle size of 0.12 μm are diluted with PBS to give a suspension having a latex concentration of 5% (w / v) in 2 m.
1 was prepared. Next, a solution (10 mg) of the anti-Alb monoclonal antibody fragmented in the above (2) diluted with PBS was used.
/ Ml) was added and mixed. After shaking at 37 ° C for 4 hours, unreacted antibodies were removed by centrifugation, and BSA was removed.
1 ml of an aqueous solution containing 5% was added. Next, 99 ml of 0.1 M sodium chloride and 0.1% sodium azide containing 0.1 M HEPES at pH 8.0 were added to prepare a suspension.

(5) Preparation of Alb test solution. 0.1 M HEPES-S, Alb antigen solution
Diluted with HEPES-S at 0.2, 0.4, 0.
8, 3.1, 6.3, 25, and 50 μg / ml were prepared as test liquids.

(6) Measurement method. 5 μl of the test solution was added to 300 μl of the above suspension in a plastic cell, and the mixture was stirred.
The change in absorbance at nm was measured. For the above operation, an automatic analyzer Hitachi 7070 was used.

(7) Relationship with change in absorbance. Table 2 shows the results of the measurement of the Alb test solution with the prepared reagent.
And FIG.

Comparative Example 2 (1) Preparation of latex suspension carrying anti-human Alb antibody. Polystyrene particles having an average particle size of 0.12 μm
To prepare a 2 ml suspension having a latex concentration of 5% (w / v). Then, a solution (10 mg / ml) of monoclonal antibodies A and B of anti-Alb diluted with PBS was added to 1
ml was added and mixed. After shaking at 37 ° C. for 4 hours, unreacted antibodies were removed by centrifugation, and 1 ml of an aqueous solution containing 5% BSA was added. Then pH 8, containing 99 ml of 0.1 M sodium chloride and 0.1% sodium azide.
The suspension was prepared by adding 0.1M HEPES-S to the suspension.

(2) Preparation and measurement of Alb test solution. Preparation of the Alb test solution and measurement using the anti-human Alb antibody-carrying latex suspension were performed in the same manner as in Example 2.
The results are shown in Table 2 and FIG.

[0041]

[Table 2]

Example 3 (1) Anti-human pepsinogen II antibody (anti-human PGII
Antibody). 10 mg / ml anti-human PGII monoclonal antibody A
Prepared in PBS to a concentration of 1.
Add 5 μl of 2-mercaptoethanol (Wako Pure Chemical Industries). After 90 minutes incubation at 37 ° C, N-
Add 0.01 mg of ethylmaleimide (Sigma). After incubation at 37 ° C. for 10 minutes, Super
20 m using a dex200 (Pharmacia) column
The desired fraction can be obtained by gel filtration with M EDTA and 0.1 M phosphate buffer. This immunoglobulin fragment was used for the following preparation.

(2) Anti-human PGI fragmented by reduction treatment
Preparation of I antibody-loaded latex suspension. A polystyrene particle having an average particle diameter of 0.3 μm is diluted with PBS to give a suspension having a latex concentration of 5% (w / v) in 2 ml.
Prepared. Next, the anti-human PGI fragmented in the above (1)
I monoclonal antibody diluted with PBS (10m
g / ml) was added and mixed. After shaking at 37 ° C. for 4 hours, unreacted antibodies were removed by centrifugation, and BS
1 ml of an aqueous solution containing 5% of A was added. Then 99ml
And 0.1 M HEPES at pH 8.0 containing 0.1 M sodium chloride and 0.1% sodium azide.

(3) Preparation of PGII test solution 0.1 M PBS and PGII antigen solution in 0.1 M PBS
0,1,3,10,30,100ng / m
1 and used as a test solution.

(4) Measurement method. Reagent volume: 300 μl, sample volume: 10 μl, measurement wavelength 54
Under the condition of 6 nm, the automatic analyzer Hitachi 7 was used in the same manner as in Example 1.
070 was used to measure the change in absorbance.

(5) Relationship with change in absorbance The PGII test solution was measured using the prepared reagent.

Comparative Example 3 A latex suspension was prepared in the same manner as in Example 3 using untreated monoclonal antibodies A and B of anti-PGII.
Using this suspension, a PGII test solution was measured under the same conditions as in Example 3. Table 3 shows the results together with the results of Example 3.
And FIG.

[0048]

[Table 3]

Example 4 (1) An anti-alpha fetoprotein antibody (anti-AFP antibody) as a polyclonal antibody was treated under the same conditions as in Example 2 to obtain a fragmented antibody (Facb). Using this antibody fragment and an untreated anti-AFP antibody,
A latex suspension was prepared in the same process as in Example 1 to obtain a latex suspension carrying an anti-AFP antibody Facb antibody and a latex suspension carrying an anti-AFP antibody.

(2) Preparation of AFP test solution. 0.1 M PBS, AFP antigen solution is diluted with 0.1 M PBS, and 0, 1, 3, 10, 30, 100, 300,
The test solution was prepared at 1000 ng / ml.

(4) Measurement method. Except for setting the sample volume to 20 μl, the absorbance change of the AFP test solution was measured in the same manner as in Example 1. The results are shown in Table 4 and FIG.

[0052]

[Table 4]

As shown in Tables 1 to 4, in all cases, the fragmentation treatment showed that the reactivity was improved in a low concentration range. Also, even in the high concentration range,
Has sufficient reactivity. In addition, nonspecific agglutination when measuring serum did not occur in any of the reagents of Examples 1 to 4 and Comparative Examples 1 to 4.

Example 5 (1) Molecular weight of fragmented antibody The latex supporting the anti-PSA fragmented antibody, collected by centrifugation, is brought into contact with a surfactant (Triton-X or the like) to release the supported antibody. This solution was used as a sample for the Phast-system
The molecular weight was confirmed by SDS-PAGE (Pharmacia).

As a result of SDS-PAGE, a single band was confirmed at a molecular weight of about 60,000 slightly below albumin.
In addition, no band could be confirmed around 150,000, which is the molecular weight of IgG.

[0056]

As described above, by carrying an antibody fragment by a reduction treatment on an insoluble carrier, the reactivity was improved in both the low concentration range and the high concentration range, and an immunoagglutination reagent with improved linearity was obtained.

[Brief description of the drawings]

FIG. 1. Calibration curve of fragmented ALB antibody-carrying reagent

FIG. 2: Calibration curve of half molecule anti-PGII antibody-carrying reagent

FIG. 3 Calibration curve of fragmented anti-AFP antibody-carrying reagent

Claims (7)

[Claims] 1. An immunological agglutination reagent comprising an antibody molecule fragmented with a reducing agent or an antibody fragment obtained by treatment with a reducing agent. 2. The immunological agglutination reagent according to claim 1, wherein the immunological agglutination is latex agglutination. 3. The method for producing an immunological agglutination reagent according to claim 1, comprising the following steps: 1) treating an antibody with a reducing agent to obtain an antibody fragment; 2) The antibody of 1) is carried on an insoluble carrier. 4. The immunological agglutination reagent according to claim 1, wherein the fragmented antibody molecule or antibody fragment is a half-molecule antibody, Facb-converted half-molecule antibody, or Facb-half-molecule. 5. The immunological agglutination reaction according to claim 1, wherein the fragmented antibody molecule or antibody fragment is reduced by any of 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, and dithiothreitol. reagent. 6. The immunological agglutination reagent according to claim 1, wherein the method for supporting the fragmented antibody molecule or antibody fragment on a carrier is physical adsorption or chemical bonding. 7. An immunological measurement method using the immunological agglutination reagent according to claim 1.