JP4178514B2 - Antibody screening method - Google Patents

Description

【００３０】
【発明の効果】
本発明により、簡便に抗体をスクリーニングして、アフィニティの高い抗体を選択することが可能である。同一の抗原に対する複数の抗体は金属基板上に固定されてアレイが作製され、表面プラズモン共鳴法によってラベルフリーに抗原への結合挙動が観察され、平衡定数が容易に得られる。
【図面の簡単な説明】
【図１】実施例に使用したフォトマスクパターン。黒色部分にクロムがコーティングされ光を遮断する。
【図２】実施例１で使用したスポッティングのパターン。上図がスライド上を表し、下図が９６穴プレートを示す。９６穴プレートのＡ１〜Ｂ１２のウェル内のサンプルを、上図のパターンでスポットした。実施例１ではＢ２〜１２はブランクとした。
【図３】実施例で使用したピンの先端の概略図
【図４】実施例で使用したピンの取り付けヘッド内部の概略図
【図５】実施例の自動スポッターの内部構造概略
【図６】実施例でのスライド上の反応スキーム
【図７】実施例での抗体を固定化したスライドのＳＰＲイメージング像
【図８】実施例での抗原の濃度を段階的に増加させた場合のＳＰＲシグナル変化
【図９】図８のデータから作成したスキャッチャードプロット[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method of selecting a high affinity antibody or a combination of antibodies by a surface plasmon resonance method using an antibody array.
[Prior art]
Monoclonal antibodies are used in a very wide range and are applied from basic research to pharmaceuticals. Monoclonal antibodies are generally obtained by fusing spleen cells and myeloma cells of mice immunized with an antigen to produce hybridoma cells and purifying them from the culture supernatant. When hybridoma cells are established, it is necessary to screen by assaying the monoclonal antibodies produced by each clone in order to select strains that produce excellent antibodies with high affinity.
[0003]
As a conventional method, the ELISA method has been often used. Although this method has already been established, the operation is complicated, and when many samples are evaluated by changing the concentration of the antibody, the number of measurements becomes extremely large, which is very troublesome.
[0004]
On the other hand, methods for measuring antigen-antibody reaction using surface plasmon resonance (SPR) and ellipsometry, which are analysis techniques that do not require labeling, using an antibody immobilized on a metal substrate surface are disclosed and known (for example, patents). Reference 1 and 2). However, these disclosures only show that the SPR method is used for individual antigen-antibody reactions, and do not show how to efficiently select antibodies with high affinity for the antigen. That is, there is no disclosure of an example in which a plurality of types of antibodies having the same antigen as in the present invention are prepared and antibodies are screened using an array in which the antibodies are immobilized on a metal substrate. The present invention discloses a method of screening antibodies using an array of antibodies produced on a metal substrate.
[0005]
[Patent Document 1]
US Pat. No. 5,629,213 [Patent Document 2]
International Publication No. WO00 / 04382 Pamphlet [0006]
[Problems to be solved by the invention]
An object of the present invention is to use an array in which antibodies are immobilized on a metal substrate and screen them by the SPR method to efficiently select antibodies with high affinity. This technique establishes a hybridoma strain that produces monoclonal antibodies. It can be used for occasions.
[0007]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. The amino group of a plurality of antibodies having the same antigen molecule is reacted with a functional group on a flat glass substrate covered with a thin gold layer to immobilize the antibody on the surface , and the remaining functional group has an amino group. Using an array where the terminal polyethylene glycol was reacted to perform a blocking reaction ,
The concentration (C) of the antigen was stepwise doubled from 1 μg / ml to 8 μg / ml, and the signal at equilibrium (Req) at each concentration was measured by surface plasmon resonance imaging ,
The binding equilibrium constant of the antigen-antibody reaction is calculated from the slope of the straight line obtained by plotting Req / C and C (scatchard plot method),
A method for screening an antibody, comprising selecting an antibody having a high affinity .
2. 2. The antibody screening method according to 1, wherein the antibody is a monoclonal antibody or a mixture of a plurality of monoclonal antibodies .
3. 3. The antibody screening method according to 1 or 2, wherein the amino group-terminated polyethylene glycol is an amino group-terminated polyethylene glycol having a molecular weight of 2,000.
4). Functional group is a succinimide group by amine coupling method, the screening method of an antibody as claimed in any one of one to 3, characterized in that the antibody is immobilized on the surface.
5. 5. The antibody screening method according to 4, wherein a succinimide group is introduced onto a flat glass substrate covered with a thin gold layer by the following methods (i) to (iv).
(I) A step of bonding polyethylene glycol (PEG thiol) having a thiol group at the terminal to the entire flat glass substrate covered with a thin gold layer
(Ii) A step of placing a photomask, irradiating with an ultra-high pressure mercury lamp, and removing PEG thiol in the irradiated portion
(Iii) Step of introducing a carboxyl group into the irradiated part
(Iv) A step of activating the carboxyl group as a succinimide ester
6). 6. The antibody screening method according to 5, wherein the end of PEG thiol is a methoxy group.
7). The method for screening an antibody according to claim 5 or 6, wherein the molecular weight of PEG thiol is 5,000.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below. The present invention discloses a method for screening antibodies using an array in which a plurality of antibodies are immobilized on the same metal substrate.
[0009]
In the present invention, there are a plurality of antibodies to be screened and the same molecules are used as antigens. However, the epitope is not limited to a single or the same. That is, the antibody may be a monoclonal antibody or a polyclonal antibody, or a combination of monoclonal antibodies having different epitopes. Different combinations / mixing ratios are also treated as different antibodies because of different affinity. A plurality of antibodies are immobilized on one metal substrate to form an array.
[0010]
In the measurement at the time of screening, a solution containing an antigen is exposed on a substrate, the antigen is trapped by an antigen-antibody reaction, and the trapped state is observed.
The SPR method is preferable as a means for observing the adsorption of the antigen on the surface. SPR is a label-free interaction analysis method, and is preferable because it is not necessary to label an antigen with a fluorescent substance or a radioisotope. Further, since the SPR signal can be observed in real time, the solution containing the antigen can be continuously flowed on the sensor surface until the interaction is saturated. In addition, since this is a semi-quantitative detection method, a change in signal corresponds to the amount adsorbed, so that it is possible to perform kinetic analysis, which is preferable.
Kinetics analysis provides an equilibrium constant for the antigen-antibody reaction, making it possible to select antibodies with high affinity.
[0011]
The maximum number of screenings that can be performed at one time is the number that can be observed by SPR, that is, the number of spots in the array. The number of spots in the array is preferably 6 or more, more preferably 12 or more, and still more preferably 20 or more. Further, the upper limit of the number of spots is not limited as long as observation can be performed, but is approximately 10,000.
A plurality of types, preferably 4 types or more, and more preferably 6 types or more antibodies are spotted on these arrays. One spot may be provided for one kind of antibody, and it is also preferable to increase the number of data by spotting at multiple places.
[0012]
Gold is preferable as the metal of the metal substrate. This is because gold can be observed by SPR and it is easy to introduce a functional group on the surface by a gold-sulfur bond. The metal substrate is preferably a flat glass substrate coated with a thin gold layer. Glasses having various refractive indexes are prepared, and it is preferable because a slide can be easily obtained by cutting. In addition, a flat substrate is preferable because molding is easy and array fabrication is easy.
[0013]
As the SPR method, an SPR imaging method capable of observing SPR signal changes in a wide range is preferable. The SPR imaging method is a method in which a polarized light beam is irradiated onto a glass substrate coated with a thin metal layer, and the reflected image is passed through a wavelength filter and then photographed with a CCD camera or the like.
[0014]
Thus, an antigen-antibody reaction is observed, and its kinetics value is analyzed from the SPR signal change. In the case of interaction with low affinity, dissociation is observed by flowing a buffer solution after interaction, and the dissociation rate constant can be calculated from the rate of dissociation. However, in the case of an antigen-antibody reaction, affinity is high and it is difficult to analyze dissociation. Therefore, the equilibrium constant is preferably calculated by the Scatchard plot method. In this method, the concentration (C) is increased stepwise, and the equilibrium value Req of the signal at that concentration is obtained. Plotting Req / C and Req, the equilibrium constant is obtained from the slope.
[0015]
Various means can be mentioned as means for immobilizing the antibody. An amino coupling method in which a carboxyl group on the surface is activated with water-soluble carbodiimide and N-hydroxysuccinimide and reacted with an amino group in an antibody is easy and preferable. Further, the antibody can be reacted with a maleimide group and a disulfide group formed on the surface by thiol coupling as F ′ (ab ′) ₂ by digestion with pepsin and as Fab ′ by reduction. Further, a means for immobilization using a sugar chain contained in an antibody is also conceivable.
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
[0016]
[Example]
Polyethylene glycol having a thiol group at the end (PEG thiol: SUNBRIGHT MESH-50H manufactured by NOF Corporation) was dissolved in a mixed solution of 7 ml of ethanol: water = 6: 1 at a concentration of 1 mM. PEG thiol has a molecular weight of 5,000 and is very hydrophilic. Moreover, the terminal of PEG is a methoxy group and has almost no reactivity.
[0017]
A gold-deposited slide obtained by depositing 3 nm of chromium on a Lak10 glass slide of 18 mm square and 1 mm thickness and depositing 45 nm of gold was immersed in the PEG thiol solution for 3 hours to bond PEG thiol to the entire gold substrate.
[0018]
After washing the slide with Milli-Q water and ethanol and drying by air spray, place the photomask shown in Fig. 1 on this slide and irradiate it with a 500W ultra high pressure mercury lamp (USHIO) for 2 hours. The PEG thiol was removed from the portion. The photomask pattern has 96 squares of 500 μm × 500 μm arranged.
[0019]
After washing with Milli-Q water and ethanol, the slide was immersed in a 1 mM solution of 7-carboxyl-1-heptanethiol (7-CHT: Dojindo Laboratories) for 1 hour to introduce a carboxyl group into the ultraviolet irradiation part.
[0020]
300 μL of 0.2 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (water-soluble carbodiimide) /0.05 M N-hydroxysuccinimide PBS (−) solution was dropped on the slide and reacted for 1 hour. And the COOH group was activated as a succinimide ester.
[0021]
The slide was washed with water, dried by air spraying, and mounted on an automatic spotter. Thirteen types of anti-goat IgG monoclonal antibodies were adjusted to a concentration of 100 μg / ml (PBS (−)), and 10 μl was prepared in wells A1 to 12 and B1 of a 96-well plate. Spotting was performed with n = 4 while maintaining the internal humidity of the automatic spotter at 81 ± 2% and a temperature of 25 ° C. Humidity was measured by placing an AS-1 NT-1800 digital thermohygrometer next to the chip yard.
[0022]
A spotting pattern is shown in FIG. In this way, spotting was performed in a manner that reflected how the sample was placed in the 96-well plate. Since the B2-12 wells do not contain a monoclonal antibody sample, they become blank spots.
[0023]
A view of the tip of the pin used for spotting is shown in FIG. The pin has a diameter of 487 μm and has a groove with a width of 200 μm. Accordingly, the area is 0.09 mm ² . FIG. 4 shows a schematic diagram of the internal structure of the head portion to which the pins are attached. The pin is attached to an internal pin fixing jig through a guide, and the pin fixing jig is movable in the vertical direction. Soft urethane foam is provided as a cushioning material on the pin fixing jig. When the pin is pressed against the substrate, the pin moves upward, the cushioning material is compressed, the shock is reduced, and moderate stress is applied to the pin. give.
[0024]
Spotting is done automatically. FIG. 5 shows an outline of the internal arrangement of the automatic spotter used. The pins are immersed in a 96-well plate, holding the sample in the well, moving onto the slide and spotting. In the case of the embodiment, n = 4, so that the 96-hole plate and the slide are reciprocated three times. At the end of the fourth spotting, the pin moves to the wash bath and is washed twice with MilliQ water. After washing, the vertical movement is performed 3 times with a swing width of 30 mm in the dryer, and the pins are dried. The sample is taken in the next well, and the same operation is automatically performed under the control of the attached computer.
The humidity was adjusted by blowing a mist generated by ultrasonic waves into the spotter, and the internal humidity was measured by measuring the humidity around the chip place and switching the ultrasonic on / off based on this data.
[0025]
After the spotting was completed, the slide was taken out from the spotter, immediately moved into the humidified chamber, held for 2 hours, the functional group on the slide reacted with the amino group of the antibody, and the antibody was immobilized on the surface. The humidification chamber has a structure in which water is applied to the bottom of the container and a slide can be placed in the middle stage, and since it can be sealed, the reaction can be continued without drying for a long time. The internal humidity is almost 100%.
After the completion of the reaction, the chip was washed several times with milli-Q water, water was removed by air spraying, and an amino group-terminated polyethylene glycol aqueous solution having a molecular weight of 2,000 was adjusted to pH 8.5 at a concentration of 2 mg / ml, and 300 μl Was poured onto the slide, and the remaining succinimide group was reacted with polyethylene glycol to perform a blocking reaction. The reaction scheme for surface immobilization is shown in FIG.
[0026]
The slide on which the antibody was immobilized was washed with water three times, dried by air spraying, and set in an SPR imaging apparatus (manufactured by Toyobo). PBS (−) was passed as a buffer solution at a flow rate of 250 μl / ml for 30 minutes, and then stabilized at a flow rate of 100 μl / ml for 30 minutes. FIG. 7 shows an SPR imaging image at that time. The antibody is immobilized on the spot portion that is white, and is not immobilized on the other portions. No scratches were found at the spot where the sample solution was spotted due to contact with the pin.
[0027]
Next, goat IgG, which is an antigen, was flowed in a stepwise doubling of the concentration (C) from 1 μg / ml to 8 μg / ml, and a signal (Req) at equilibrium at each concentration was obtained. The change of the SPR signal at this time is shown in FIG.
[0028]
FIG. 9 shows the results of plotting Req / C and C (Scatchard plot). In almost all cases, a linear relationship is obtained. The binding equilibrium constant is calculated from the slope of the straight line in FIG. However, data whose correlation coefficient is less than 0.8 due to low signal is omitted.
Thus, the antibody was immobilized on the metal substrate, and an antibody with high affinity could be selected.
[0029]
[Table 1]

[0030]
【The invention's effect】
According to the present invention, it is possible to simply screen antibodies and select antibodies with high affinity. A plurality of antibodies against the same antigen are immobilized on a metal substrate to produce an array, and the binding behavior to the antigen is observed in a label-free manner by the surface plasmon resonance method, and the equilibrium constant can be easily obtained.
[Brief description of the drawings]
FIG. 1 is a photomask pattern used in Examples. Black part is coated with chrome to block light.
2 shows a spotting pattern used in Example 1. FIG. The upper figure shows the top of the slide, and the lower figure shows the 96-well plate. Samples in wells A1 to B12 of a 96-well plate were spotted in the pattern shown above. In Example 1, B2 to 12 were blank.
FIG. 3 is a schematic view of the tip of a pin used in the embodiment. FIG. 4 is a schematic view of the inside of the pin mounting head used in the embodiment. FIG. 5 is a schematic view of the internal structure of the automatic spotter of the embodiment. Reaction Scheme on Slide in Example [FIG. 7] SPR Imaging Image of Slide Immobilized with Antibody in Example [FIG. 8] Change in SPR Signal when Stepwise Increases Antigen Concentration in Example 9 is a Scatchard plot created from the data of FIG.

Claims (7)

The amino group of a plurality of antibodies having the same antigen molecule is reacted with a functional group on a flat glass substrate covered with a thin gold layer to immobilize the antibody on the surface , and the remaining functional group has an amino group. Using an array where the terminal polyethylene glycol was reacted to perform a blocking reaction ,
The concentration (C) of the antigen was stepwise doubled from 1 μg / ml to 8 μg / ml, and the signal at equilibrium (Req) at each concentration was measured by surface plasmon resonance imaging ,
The binding equilibrium constant of the antigen-antibody reaction is calculated from the slope of the straight line obtained by plotting Req / C and C (scatchard plot method),
A method for screening an antibody, comprising selecting an antibody having a high affinity . The antibody screening method according to claim 1 , wherein the antibody is a monoclonal antibody or a mixture of a plurality of monoclonal antibodies . 3. The antibody screening method according to claim 1 or 2, wherein the amino group-terminated polyethylene glycol is an amino group-terminated polyethylene glycol having a molecular weight of 2,000. Functional group is a succinimide group by amine coupling method, the screening method of an antibody according to any one of claims 1 to 3, characterized in that the antibody is immobilized on the surface. The antibody screening method according to claim 4, wherein a succinimide group is introduced onto a flat glass substrate covered with a thin gold layer by the following methods (i) to (iv).
(I) A step of bonding polyethylene glycol (PEG thiol) having a thiol group at the terminal to the entire flat glass substrate covered with a thin gold layer.
(Ii) A step of placing a photomask, irradiating with an ultra-high pressure mercury lamp, and removing PEG thiol in the irradiated part
(Iii) Step of introducing a carboxyl group into the irradiated part
(Iv) A step of activating the carboxyl group as a succinimide ester The method for screening an antibody according to claim 5, wherein the terminal of PEG thiol is a methoxy group. 7. The antibody screening method according to claim 5 or 6, wherein the molecular weight of PEG thiol is 5,000.

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