JP2010164551A - Quick screening method of material having intermolecular interaction using fluorescence polarization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of screening a material having intermolecular interaction by fluorescence polarization measurement by suppressing rise of a background, even when a material having self-fluorescence is included in an object. <P>SOLUTION: In this screening method of the material having intermolecular interaction to a labeled substance in an object sample by measuring fluorescence polarization from the fluorescence labeled substance, an excitation wavelength (λ1) suppressing self-fluorescence of the object sample at a fluorescence measuring wavelength (λ2) is used. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for rapidly screening a substance having an intermolecular interaction using a fluorescence polarization method.

  When screening a monoclonal antibody that binds to an antigen (a hybridoma that produces the monoclonal antibody), an ELISA is often used. As an example, a hybridoma culture supernatant (a monoclonal antibody produced by the hybridoma) is reacted with an ELISA plate on which the antigen is immobilized to form an antigen-antibody complex. Thereafter, the unbound antibody is separated, an enzyme-labeled antibody that reacts specifically with the antibody is bound, an unreacted enzyme-labeled antibody is separated, and then a substrate of the enzyme as a label is added. Thereby, a monoclonal antibody that binds to the antigen can be screened, or a hybridoma that produces the monoclonal antibody can be screened.

  While the above method can confirm the presence of an antibody with high sensitivity, an operation to separate unreacted substances (so-called B / F separation operation) is required several times before the result is obtained. As the number of types (number) increases, a very large number of man-hours are required.

  For example, screening methods that do not require B / F separation, such as FRET (Fluorescence Resonance Energy Transfer), TR-FRET (Time Resolved FRET), Alphascreen (trade name), or fluorescence polarization are also known. In fact, in the methods such as FRET, TR-FRET, and Alphascreen (trade name), dedicated reagent kits for carrying out these methods are also commercially available. However, such a reagent is extremely expensive, and it is not practical to use it for the purpose of processing a large number of samples like screening of a monoclonal antibody.

  On the other hand, the fluorescence polarization method introduced by Perrin (Non-Patent Document 1) is excellent in operability because it is only necessary to label an antigen with a fluorescent substance, and it can keep running costs low. Suitable for screening to be processed. When a fluorescent molecule in a liquid is excited by plane polarized light, it emits fluorescence at the same polarization plane if the fluorescent molecule does not move. However, when the fluorescent molecule moves such as rotating, fluorescence is emitted to a plane different from the excitation plane, and the fluorescence polarization is canceled. The movement of the molecule is influenced by the size of the molecule. When the fluorescent molecule is a low molecule, the movement speed is fast, so that the polarization of the emitted light is canceled and the degree of fluorescence polarization is small. On the other hand, when the fluorescent molecule is a polymer, there is almost no movement of the molecule during the excited state, and the emitted light cannot be depolarized and gives a large degree of fluorescence polarization. In the case of a fluorescently labeled low-molecular antigen, the fluorescence polarization is canceled, but when the antibody is bound to it, the molecular weight is apparently increased, and the fluorescence polarization cannot be canceled and a large fluorescence polarization degree is given. That is, by measuring the degree of fluorescence polarization, it is possible to confirm whether the antigen is bound to the antibody without performing B / F separation.

Perrin, J.M. Phys. Rad. 1, 390, 1926

  As described above, the fluorescence polarization method is suitable for monoclonal antibody screening in terms of its operability and running cost, but there are still points to be improved. First, it is possible to perform screening using a solution containing fetal bovine serum as a sample. In general, hybridomas are cultured in a medium containing about 10% fetal bovine serum, and screening is performed for monoclonal antibodies released to the medium. When a sample containing fetal bovine serum is used as a target, the background becomes high due to autofluorescence of fetal bovine serum, making screening difficult.

  Next, there is a problem of a lid of a holding container that holds a target sample for fluorescence measurement. When cultivating hybridomas, in order to avoid contamination with bacteria and the like, the plate as a holding container is covered with a lid, and the lid is not opened except for a clean bench. However, in the case of fluorescence polarization, since excitation light is usually irradiated from the top of the plate and the resulting fluorescence is measured from the top, the lid of the plate is opened for measurement, which increases the risk of contamination. Although it is conceivable to transfer the target sample from the plate for culturing to another holding container, such a method adds a complicated operation to screening by measurement of fluorescence polarization, so that the efficiency cannot be achieved. is there.

  Therefore, the present invention is a fluorescence polarization measurement that can suppress an increase in background due to autofluorescence even when the target sample contains fetal bovine serum commonly used in culturing hybridomas. It is an object of the present invention to provide a screening method for substances having intermolecular interactions such as antibodies. Another object of the present invention is to provide a screening method for a substance having an intermolecular interaction such as an antibody by fluorescence polarization measurement, which can avoid the risk of contamination by bacteria during fluorescence measurement. .

  The present invention made in view of the above problems is a screening method for a substance having an intermolecular interaction with a labeled substance in a target sample by measuring the fluorescence polarization from the fluorescent labeled substance, the fluorescence measurement An excitation wavelength (λ1) that can suppress autofluorescence of the target sample at the wavelength (λ2) is used. In addition, the present invention is characterized in that fluorescence measurement from a target sample is performed with a quartz glass lid on the target sample holding container. Hereinafter, the present invention will be described in detail.

  In the present invention, a commercially available fluorescent substance such as tetramethylrhodamine or Alexa Fluor (manufactured by Invitrogen) can be used as a fluorescent label. A fluorescent label is obtained by binding a fluorescent label to one of a pair of substances having an intermolecular interaction to be screened. The binding method is not limited as long as the substance can maintain the function as a fluorescent label. It may be a bond, a physical bond, or a bond via an affinity substance such as avidin and biotin. Substances having intermolecular interactions include, for example, antigens and antibodies, DNA (RNA) fragments and DNA (RNA) fragments or ligands and receptors as described above, and the molecular weight is within the range of symmetry of fluorescence polarization measurement. If there is no particular limitation. The case where the substance having intermolecular interaction is an antigen and an antibody will be described in detail. From the principle of measurement of fluorescence polarization, an antigen to which a fluorescent label is bound (the difference in molecular weight from the antibody is preferable in terms of measurement principle) is fluorescent. By using it as a label, it can be measured with high sensitivity. Note that the method of knowing whether or not a substance having an intermolecular interaction with a fluorescent label is present in a target sample by measuring fluorescence polarization is already a well-known method, and is described in detail in this specification. However, the principle is as described above.

  Fluorescence emitted from various substances existing in nature is generally weak in a long wavelength region. According to the knowledge of the present inventor, the combination of the excitation wavelength (λ1) and the fluorescence measurement wavelength (λ2) that suppresses the autofluorescence is also specific for the autofluorescence produced by the medium containing fetal bovine serum for hybridoma culture. Includes an excitation wavelength (λ1) near 555 nm, a fluorescence measurement wavelength (λ2) 565 to 620 nm (hereinafter referred to as a first combination), an excitation wavelength (λ1) near 650 nm, and a fluorescence measurement wavelength (λ2) 660 to 720 nm. Can be exemplified (hereinafter referred to as the second combination). In the first combination and the second combination, autofluorescence from a medium containing fetal bovine serum for hybridoma culture can be suppressed to a low level, so that the object of the present invention can be achieved. Regarding the excitation wavelength, autofluorescence from the medium containing fetal bovine serum can be sufficiently suppressed even at wavelengths slightly deviated from the respective wavelengths. Moreover, it is technically difficult to prepare excitation light that does not contain any light of other wavelengths. Therefore, the vicinity of 555 nm (650 nm) in the present invention means that light of 555 nm (650 nm) is included or light having a wavelength of near 555 nm (650 nm) is used.

  As a suitable fluorescent label for carrying out the first combination or the second combination relating to the excitation wavelength and the fluorescence measurement wavelength, tetramethylrhodamine can be exemplified for the first combination, and the second combination is exemplified. May be Alexa Fluor 647 (Invitrogen) or Alexa Fluor 680 (Invitrogen). Tetramethylrhodamine has a maximum absorption wavelength of 555 nm. Even at a wavelength slightly deviated from the wavelength, tetramethylrhodamine can be sufficiently excited with fluorescence although the efficiency is lowered. Similarly, Alexa Fluor 647 has a maximum absorption wavelength of 650 nm, and Alexa Fluor 680 has a maximum absorption wavelength of 679 nm. Even at a wavelength slightly deviated from the wavelength, the fluorescence can be sufficiently excited although the efficiency is lowered. . Therefore, it is not strictly necessary to use excitation light of 555 nm, 650 nm, or 679 nm, and excitation light having a wavelength in the vicinity thereof may be used. In addition, since it is technically difficult to prepare excitation light that does not include light of any other wavelength, for example, when excitation is performed at 555 nm, the bandpass width of the light source of the fluorescent device is 20 nm around 555 nm. Usually, a filter having a wavelength of about 535 nm to 575 nm is used as excitation light. Therefore, when the fluorescent label is excited in the present invention, excitation light including light having the maximum absorption wavelength or light having a wavelength near the maximum absorption wavelength may be used.

  Whether the monoclonal antibody is present in the cell culture medium containing fetal bovine serum in the first or second combination of the excitation wavelength (λ1) and the fluorescence measurement wavelength (λ2) suitable for practicing the present invention Can be determined. Even when a substance that emits autofluorescence other than fetal bovine serum is contained, interference with measurement of fluorescence polarization is mainly caused by fetal bovine serum, as in the target sample used in Examples described later. When it is derived from the emitted autofluorescence, it is possible to carry out good fluorescence polarization measurement (screening) by combining the excitation wavelength (λ1) and the fluorescence measurement wavelength (λ2) as described above. .

  When a substance that emits autofluorescence (other than fetal bovine serum) that is not normally used is added to the target sample and used, the excitation wavelength (λ1) and fluorescence measurement wavelength (λ2) described above are used. In a specific combination of (), it is not always possible to carry out good fluorescence polarization measurement (screening). In such a case, by using the method described in the examples of the present invention, a combination of an excitation wavelength (λ1) and a fluorescence measurement wavelength (λ2) suitable for carrying out the present invention for the target sample, and A suitable fluorescent label for carrying out the present invention in the combination may be appropriately determined.

  In the measurement of fluorescence polarization in the present invention, it is possible to avoid the risk of contamination due to bacteria or the like during fluorescence measurement by covering the preparation of the target sample with a cover glass cover. In particular, in the case of a quartz glass plate with better optical characteristics than a normal cover glass, according to the knowledge of the present inventor, even if the thickness is about 1 mm, the measurement result does not vary depending on the presence or absence of a lid and the same result is obtained be able to.

  According to the present invention, even if the target sample contains a substance that emits autofluorescence such as fetal bovine serum that is commonly used in culturing hybridomas, the background due to the autofluorescence is increased. It is possible to provide a screening method with high sensitivity for substances having intermolecular interactions such as antibodies by fluorescence polarization measurement. In addition, since the present invention can be carried out in a state where the container holding the target sample is covered, it is possible to avoid the risk of contamination due to bacteria or the like during fluorescence measurement. It is.

FIG. 1 shows the results of Example 1. FIG. 2 shows the results of Example 2. FIG. 3 shows the results of Example 3 when Marina Blue was used. FIG. 4 shows the results when Alexa Fluor 430 (manufactured by Invitrogen) is used in Example 3. FIG. 5 shows the results when fluorescein (FITC) was used in Example 3. FIG. 6 shows the results when tetramethylrhodamine was used in Example 3. FIG. 7 shows the results when Alexa Fluor 594 (manufactured by Invitrogen) is used in Example 3. FIG. 8 shows the results when Alexa Fluor 647 (manufactured by Invitrogen) was used in Example 3. FIG. 9 shows the results of Example 4. FIG. 10 shows the results of Example 5. FIG. 11 shows the results of Example 6. FIG. 12 shows the results of Example 7. FIG. 13 shows the results of Example 8. FIG. 14 shows the results of Example 8. FIG. 15 shows the results of Example 9. FIG. 16 shows the results of Example 10. FIG. 17 shows the results of Example 10. FIG. 18 shows the results of Example 11. FIG. 19 shows the results of Example 12. FIG. 20 shows the results of Example 13.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these Examples do not limit this invention.

Example 1
The fluorescence spectrum was measured when PBS and a commercially available medium for animal cell culture (trade name: GIT, manufactured by Nippon Pharmaceutical Co., Ltd.) were excited with the excitation wavelength (λ1, 488 nm) of fluorescent labeling fluorescein. . The results are shown in FIG. Although almost no fluorescence was observed with PBS, autofluorescence of various components contained in the culture medium was observed (solid line in FIG. 1).

  The same amount of fluorescein (FITC) is added to each target sample, fluorescence is excited at the excitation wavelength (λ1) of fluorescein, and fluorescence is measured at the fluorescence measurement wavelength (maximum fluorescence wavelength) (λ2, 518 nm) When the S / N ratio was calculated, it was 11 in the case of PBS but 2.2 in the case of the culture medium. Therefore, when fluorescence excitation is performed at the excitation wavelength (λ1) of fluorescein and fluorescence measurement is performed at the fluorescence measurement wavelength (λ2), the S / N ratio is lowered due to autofluorescence of the culture medium, and good fluorescence polarization is achieved. Is difficult to measure.

Example 2
After mixing a certain amount of fluorescein with anti-fluorescein antibody solutions prepared at various concentrations, these were added to PBS or E-RDF serum-free medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) to be used as a target. It measured as a sample using the commercially available measuring apparatus (Brand name: BEACON, Takara Shuzo Co., Ltd. product). The results are shown in FIG. FIG. 2 shows that when the E-RDF serum-free medium is used as a target sample, the detection sensitivity is reduced to about 1/10 compared with PBS. This is considered to be the influence of the components in the medium. For E-RDF serum-free medium used in normal cell culture, 10% fetal bovine serum was added, and the effect of autofluorescence was so great that measurement could not be performed.

Example 3
Using the above-described commercially available medium for animal cell culture (trade name: GIT) as a target, various fluorescent substances were used as fluorescent labels, and fluorescence spectra were examined when they were excited at their excitation wavelengths (λ1). The results are shown in FIGS. In each graph, the fluorescence intensity value obtained at the excitation wavelength (λ1) of fluorescein is indicated by a broken line, but the fluorescence measurement wavelength (λ2) is within a range indicating a value lower than the value indicated by the wavy line. If it has a fluorescent label, it is highly possible that fluorescence polarization can be measured with higher sensitivity than fluorescein. From the results of this example, the possibility was confirmed with tetramitylrhodamine (λ1; 555 nm, λ2; 580 nm, FIG. 6) and Alexa Fluor 647 (Invitrogen) (λ1; 650 nm, λ2; 668 nm, FIG. 8). It is done.

Example 4
The fluorescence of PBS or a commercially available animal cell culture medium (trade name: GIT) was measured using fluorescein or tetramethylrhodamine (hereinafter “TAMRA”). A commercially available apparatus (trade name Analyst GT, manufactured by Molecular Devices) was used for the measurement. The results are shown in FIG. From FIG. 9, when TAMRA is used, the fluorescence intensity of the medium for animal cell culture is reduced to about 1/10 compared with the case of using fluorescein, and the S / N ratio is greatly improved. I understand that

Example 5
A fluorescent label (hereinafter “E2-TAMRA”) in which estradiol (hereinafter “E2”) was bound to TAMRA was prepared as follows (see FIG. 10). First, E2-NH2 was prepared by introducing an amino group at the 6-position of E2, and reacted with TAMRA into which an NHS group was introduced (hereinafter “TAMRA-NHS”, manufactured by Molecular Devices) in DMSO. Note that TAMRA-NHS was reacted in a large excess so that unreacted E2-NH2 was not left (1 in FIG. 10). Thereafter, a large excess of TAMRA-EHS was removed using a column on which the anti-E2 antibody was immobilized, to obtain E2-TAMRA (2. in FIG. 10).

Example 6
Using E2-TAMRA prepared in Example 5, antigen-antibody reaction in a commercially available animal cell culture medium (trade name GIT) was observed. Commercially available apparatus for measuring the fluorescence polarization value of each sample with a target sample of various concentrations of anti-E2 antibody (60 microliters) plus a certain amount of E2-TAMRA (20 microliters) dissolved in the medium. (Product name: Analyst GT). The results are shown in FIG. From FIG. 11, it was confirmed that the fluorescence polarization value (mP value) increased in the medium as the antibody concentration increased. From the rule of thumb that when a hybridoma culture supernatant is used as a target sample, the monoclonal antibody is often contained at a concentration of several micrograms / mL to several tens of micrograms / mL. By applying the method of the invention, it can be seen that whether or not a monoclonal antibody is present in the culture supernatant can be measured easily and rapidly.

Example 7
The specificity of the screening method of the present invention was evaluated by the following method. 0.4 microliters of rabbit serum (anti-E2 rabbit antiserum or anti-T3 rabbit antiserum) was added to 60 microliters of a commercially available animal cell culture medium (trade name: GIT), and a certain amount dissolved in GIT. E2-TAMRA (20 microliters) was added to prepare a target sample, and the fluorescence polarization value was measured. The results are shown in FIG. As can be seen from FIG. 12, an increase in mP value was confirmed only for the anti-E2 rabbit antiserum. From this, it can be seen that the method of the present invention is an intermolecular interaction-specific (antigen-specific in this example) reaction.

Example 8
The mouse was immunized with E2-BSA in which E2 was bound to BSA, and after confirming that the antibody titer had sufficiently increased, the spleen was removed and the spleen cells were fused with myeloma cells by the PEG method. The presence of 67 hybridomas was visually confirmed, their culture supernatants were obtained, and it was determined by the method of the present invention whether or not a monoclonal antibody against E2 was present therein. The results are shown in FIG. The right end (Neg.) Of FIG. 13 is the measured value of the medium itself, and the left side (Posi. (Medium + anti-E2 antibody)) is a positive control in which an anti-E2 monoclonal antibody prepared in advance in the medium was added. It is. As a result, an increase in mP value was confirmed in several culture supernatants. Next, with respect to the above 67 culture supernatants, a commercially available reagent (trade name: BIACORE, manufactured by GE Healthcare Co., Ltd.) was used to evaluate whether or not a monoclonal antibody against E2 was present therein. In the method using the commercially available reagent, a sensor chip to which E2-NH2 is bound by an amine coupling method is used, and the amount (RU) of a substance that binds to E2 when a certain amount of culture supernatant is flowed is calculated. To do.

  FIG. 14 is a plot of mP values on the horizontal axis and RU values on the vertical axis. FIG. 14 shows that there is a correlation between the result of BIACORE and the result of the method of the present invention, and that the monoclonal antibody can be screened by the method of the present invention.

Example 9
In addition to the 96-well plate frequently used in the monoclonal antibody screening process, a 384-well plate or a 1536-well plate was used to investigate whether screening could be performed for a larger number of target samples. The results are shown in FIG. From FIG. 15, it can be seen that the present invention can be applied to all the multi-well plates tested in this example, and that the present invention is a very effective method for the production of monoclonal antibodies that require multi-analyte screening. .

Example 10
A certain amount of E2-TAMRA prepared in Example 5 was added to an animal cell culture medium containing various concentrations of anti-E2 antibody (trade name: GIT), and the well with the lid opened and a plate sheet film were covered. Each well or a well covered with a coverslip. The results are shown in FIG. As shown in FIG. 16 left, conventionally, the lid is opened to perform measurement. When the measurement was performed with a commercially available plate seal film, the measurement became difficult (in FIG. 16). On the other hand, when the lid was covered with a cover glass, there was no problem in the measurement (right side of FIG. 16). Therefore, a lid was made of a quartz glass plate (thickness 1 mm) having better optical properties than the cover glass, and a culture medium for animal cell culture (trade name: GIT) containing various concentrations of anti-E2 antibody was dispensed 384. A certain amount of E2-TAMRA prepared in Example 5 was added to the well plate for measurement, and the result was compared with the case where the lid was opened. The results are shown in FIG. From FIG. 17, it can be seen that there is no difference in the measurement results (the results are correlated) when the quartz glass lid is covered and when it is not covered.

Example 11
Alexa Fluor 680 (A20344: manufactured by Invitrogen) into which maleimide was introduced was reacted with 0.1 mg of the C-terminal amino acid (SEQ ID NO: 1) of BNP (brain natriuretic peptide) (1. in FIG. 18). Thereafter, using a column on which an anti-BNP C-terminal amino acid recognition antibody was immobilized, a large excess of Alexa Fluor 680 was removed, and SEQ ID NO: 1 labeled with Alexa Fluor 680 was obtained (2 in FIG. 18).

Example 12
Using the fluorescently labeled peptide prepared in Example 11, the antigen-antibody reaction in a commercially available animal cell culture medium (trade name: GIT) was observed. Fluorescence polarization values were obtained from Tecan Co., Ltd. using a measurement sample prepared by adding a certain amount of a fluorescently labeled peptide (5 microliters) dissolved in GIT medium to BNP C-terminal recognition antibodies (45 microliters) at various concentrations. It measured with the plate reader (brand name: M500). The excitation light filter used was 630 nm and the bandwidth was 35 nm, and the measurement filter was 720 nm and the bandwidth was 40 nm. The results are shown in FIG. From FIG. 19, it was confirmed that the fluorescence polarization value (mP value) increased in the medium as the antibody concentration increased.

Example 13
The presence or absence of antibody in the hybridoma culture supernatant was determined under the same conditions as in Example 12. 5 microliters of the same amount of fluorescently labeled peptide as in Example 12 was added to 45 microliters of the hybridoma culture supernatant to prepare a sample. The results are shown in FIG. From FIG. 20, it was confirmed that the hybridoma expressing the antibody can be detected by this method.

Claims (7)

A method for screening a substance having an intermolecular interaction with a label in the target sample by measuring fluorescence polarization from the fluorescent label, wherein autofluorescence of the target sample is measured at a fluorescence measurement wavelength (λ2). A method using an excitation wavelength (λ1) that can be suppressed. The method of claim 1, wherein the excitation wavelength (λ1) is near 555 nm and the fluorescence measurement wavelength (λ2) is 565 to 620 nm. The method according to claim 1 or 2, wherein tetramethylrhodamine is used as a fluorescent label. The method of claim 1, wherein the excitation wavelength (λ1) is in the vicinity of 650 nm and the fluorescence measurement wavelength (λ2) is from 660 to 720 nm. The method according to claim 1 or 4, wherein Alexa Fluor 647 (trade name) or Alexa Fluor 680 (trade name) is used as a fluorescent label. The method of claim 1, wherein the intermolecular interaction is an immune response. 2. The method according to claim 1, wherein fluorescence measurement from the target sample is performed with a quartz glass lid on the target sample holding container.

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