JP3784111B2 - Method for measuring antigen concentration - Google Patents

Description

【図面の簡単な説明】
【図１】この発明の測定手順を例示した模式図である。
【図２】プレートに固定したビオチン化ＶＬまたはニワトリ卵白リゾチームへのＶＨファージの結合量を示した棒グラフである。
【図３】プレートに固定したビオチン化ＶＬへのＶＨ−ファージの結合量に基づき作成したニワトリ卵白リゾチーム濃度の検量線である。
【図４】プレートに固定したビオチン化ＶＬへのアルカリホスファターゼ標識化ＶＨの結合量を示す吸光度をニワトリ卵白リゾチーム濃度毎にプロットしたグラフである。
【図５】フルオレセイン化ＶＨとローダミンＸ化ＶＬの蛍光比をニワトリ卵白リゾチーム濃度毎にプロットしたグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring an antigen concentration. More specifically, the present invention relates to a new method for measuring the antigen concentration in a specimen simply and with high sensitivity using intramolecular interactions of a single antibody (immunoglobulin). The method of the present invention can be carried out, for example, as a sandwich ELISA method, and is useful for clinical diagnosis for measuring various antigen concentrations in a biological sample (blood, urine, body fluid, etc.). In addition, the method can be widely applied to specific detection of all natural and non-natural substances that can be recognized as an antigen by an antibody, and can be used as a research or industrial sensor.
[0002]
[Prior art and its problems]
Conventionally, methods for measuring antigen concentration by various methods are known, and some methods are also used in clinical diagnosis.
Among these antigen concentration measurement methods, the so-called sandwich ELISA method (or sandwich RIA method) is particularly attracting attention. This method uses two types of epitopes that are recognized by different epitopes when measuring a certain antigen protein. The measurement is performed using a monoclonal antibody or a monoclonal antibody and a polyclonal antibody. That is, as a first step, a specimen containing an antigen is poured onto a measurement plate adsorbed with a mono / polyclonal antibody called a primary antibody and allowed to stand for a certain period of time. During this period, impurities other than the antigen bound to the antibody are removed by washing with a washing solution as a second step. Next, as a third step, a labeled antibody solution to which a reporter molecule such as an enzyme or a radioisotope is bound in advance is poured and left for a predetermined time. During this time, the labeled antibody binds to the antigen captured by the primary antibody, so the excess labeled antibody is washed away sufficiently with the washing solution, and then the amount of the reporter molecule remaining on the measurement plate is measured using an enzyme activity or a liquid scintillation counter. Thus, the amount of antigen in the specimen is estimated.
[0003]
The conventional sandwich ELISA method comprising the above operation procedure has the merit that almost all protein antigens can be measured as long as the necessary antibodies are obtained, but on the other hand, it requires at least two reactions and washings. Therefore, the measurement procedure is complicated, so that the measurement takes time and the equipment for automation is expensive.
[0004]
On the other hand, the inventors of the present invention pay attention to the phenomenon that the stability of the Fv region, which is an antigen recognition site of an antibody, changes depending on the presence or absence of binding to the antigen, A sandwich ELISA method using VL molecules is proposed.
The present invention further develops a sandwich ELISA method using single antibody VH molecules and VL molecules already proposed by the present inventors, has excellent sensitivity and reliability, and allows a simple and rapid measurement procedure. It is an object of the present invention to provide a new antigen concentration measurement method that can be used.
[0005]
[Means for Solving the Problems]
As a first means for solving the above problems, the present invention prepares a VH region polypeptide and a VL region polypeptide of an antibody that specifically recognizes an antigen, and labels one of the polypeptides with a reporter molecule. Labeled polypeptide, the other polypeptide is immobilized on a solid phase to form an immobilized polypeptide, an antigen-containing sample and the labeled polypeptide are brought into contact with the solid phase, and the labeled polypeptide bound to the immobilized polypeptide In the method for measuring the amount of the reporter molecule, an antigen concentration measurement method (first method) is provided, wherein the reporter molecule is an enzyme or a fluorescent dye.
[0006]
In this first method, the immobilized polypeptide is preferably immobilized on a solid phase via a bond between biotin or a tag sequence and avidin or streptavidin. Another preferred embodiment is that the enzyme serving as the reporter molecule is Escherichia coli alkaline phosphatase or a variant thereof, and the fluorescent dye is fluorescein or a derivative thereof.
[0007]
Furthermore, the present invention provides, as a second means, preparing a VH region polypeptide and a VL region polypeptide of an antibody that specifically recognizes an antigen, labeling the VH region polypeptide with a first reporter molecule, Is labeled with the second reporter molecule, the antigen-containing sample is mixed with the labeled VH region polypeptide and the labeled VL region polypeptide, and the amount of change in the interaction between the first reporter molecule and the second reporter molecule is measured. An antigen concentration measuring method (second method) is provided.
[0008]
One aspect of the second method is that the first reporter molecule and the second reporter molecule are different types of fluorescent dyes, and the amount of the complex is measured by the amount of fluorescence generated by energy transfer between the two reporter molecules. . In this case, the different fluorescent dyes are preferably fluorescein or a derivative thereof and rhodamine or a derivative thereof, respectively.
[0009]
In these first and second methods, the VH region polypeptide and the VL region polypeptide are used as expression products of vectors incorporating the gene sequences encoding them, or each gene sequence is separately incorporated 2 It can be prepared as an expression product of various types of vectors. When preparing an antibody polypeptide in this way, the labeled polypeptide can also be expressed as a fusion protein or chimeric protein with a reporter molecule.
[0010]
The measurement method of the present invention having the configuration as described above provides a new ELISA method that has succeeded in simplifying and speeding up the measurement procedure while having sensitivity and reliability equivalent to or higher than those of the sandwich ELISA method. is there.
Furthermore, the present invention also provides a labeled polypeptide, a set of labeled polypeptides, and an antigen concentration measurement kit containing these labeled polypeptides.
[0011]
Hereinafter, embodiments of the present invention will be described in more detail. In the following description, a VH region polypeptide may be described as VH, and a VL region polypeptide may be described as VL.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
In the method of the present invention, an antibody that specifically binds to any antigen to be measured is used. Such an antibody can be selected, for example, from a monoclonal antibody prepared by hybridoma technology. Next, polypeptide fragments of VH region and VL region are separately prepared from such antibodies. Each polypeptide may be longer or shorter than the VH region and VL region of the antibody as long as they can bind the target antigen in a state where they are paired. These polypeptides can be prepared as follows, for example, for antibodies that recognize all antigens.
[0013]
That is, a monoclonal antibody that specifically binds an arbitrary antigen is prepared by a known method (Kohler, G. and Milstein, C. Nature 256, 495-496, 1975), and a gene encoding the variable region of this antibody is obtained. A method using hybridization with a cDNA library (Sambrook, Frisch, and Maniatis, Monecular Cloning 2nd ed., 1989) or a method using PCR (larrick et al., Biochem. Biophys. Res. Commun. 160, 1250-1256 1989, Jones, S. and Bendig, M., Bio / Technology 9, 88-89, 1991), and this gene is cloned into a vector. A sequence encoding the VH and / or VL region is obtained from the recombinant vector, the fragment is subcloned into an expression vector, and the gene is expressed in a host cell, thereby obtaining the required amount of VH and / or VL. Can be obtained. In order to obtain a VH / VL coding sequence from an antibody gene, a desired sequence region may be excised with a restriction enzyme and amplified with a cloning vector, or a desired sequence may be amplified with a PCR method. When VH and / or VL are expressed in a host cell, a gene encoding any reporter molecule is also cloned into an expression vector, and VH and / or VL is expressed as a fusion protein or a chimeric protein with the reporter molecule. Can do.
[0014]
VH and / or VL can also be obtained by decomposing an antibody molecule with a proteolytic enzyme without using the above method. In this case, there is an advantage that the labor of gene cloning can be saved, although a device for purification and labeling reaction of fragments is required.
In the first method of the present invention, one of the polypeptides thus prepared is immobilized on a solid phase and the other is labeled with a reporter molecule. Both VH and VL can be used as a labeled polypeptide or as an immobilized polypeptide. Moreover, a well-known method is employable as the immobilization method and labeling method of polypeptide. As the reporter molecule, an enzyme or a fluorescent dye is used. When labeling with an enzyme (for example, alkaline phosphatase), the gene is incorporated into a vector so that VH or VL is expressed as a fusion protein with the enzyme, and a labeled polypeptide is prepared as a fusion protein with the enzyme. In addition, when a fluorescent dye (for example, fluorescein or a derivative thereof) is used, labeling is performed without affecting the antigen binding ability and Fv forming ability by selectively labeling the amino terminus of VH or VL with the fluorescent dye. Polypeptide can be prepared. However, in this invention, the preparation of each polypeptide is not limited to these methods, and any method may be used as long as the final purpose can be achieved.
[0015]
Next, an example of an operation procedure in the first method will be described with reference to FIG. In FIG. 1, VH is an enzyme (alkaline phosphatase: AP) labeled polypeptide, and VL is an immobilized polypeptide.
First, VL is fixed to the measurement plate. In order to fix the VL to the measurement plate efficiently, the VL may be purified and biotinylated. Thereby, it can be easily fixed to the measurement plate on which streptavidin is adsorbed. Alternatively, there is a method of preparing VL in such a form that an amino acid sequence capable of binding to streptavidin is bound to its carboxy terminus. In this case, the immobilization is completed only by reacting the culture supernatant with the streptavidin plate without particularly purifying VL.
[0016]
In this invention, the antigen concentration can be measured in principle only by the subsequent one-step reaction and washing.
That is, a measurement sample containing an antigen and VH-AP are simultaneously injected into a plate on which VL is fixed, mixed, and allowed to stand for a predetermined time. Since the VH-VL interaction is weak in the absence of antigen, no binding of VH-AP to the plate is seen in the absence of antigen. In contrast, in the presence of antigen, VH and VL bind tightly through the antigen, so VH-AP is immobilized on the plate, and the enzyme activity remaining on the plate after washing is measured by a known method. It shows a very good correlation with antigen concentration.
[0017]
The first measurement method of the present invention can be carried out by a combination of an immobilized polypeptide and a labeled polypeptide prepared in advance from an arbitrary antibody. Such a combination can also be configured as a kit comprising a solid phase having an immobilized polypeptide and a reagent containing the labeled polypeptide. In the case of such a kit, the same immobilized polypeptide and labeled polypeptide as in the first measurement method can be used.
[0018]
Furthermore, in this antigen measurement kit, the polypeptide may be labeled with filamentous phage. For example, when VH is labeled with this filamentous phage, the VH gene is incorporated into a vector plasmid so that VH is expressed as a fusion protein with the coat protein g3p of filamentous phage, and then the expressed VH-g3p The fusion protein is displayed on the M13 phage (VH phage). This is possible by using a known technique called phage display (McCafferty, J. et al., Nature 348, 552-554, 1990). That is, the phage can be obtained in the culture supernatant by infecting Escherichia coli having a fusion protein expression vector plasmid with M13 phage and culturing the bacterium under culture conditions that induce the expression of the fusion protein. In addition, the phage itself can be previously labeled with an enzyme or a dye.
[0019]
In addition, a standard solution containing various known concentrations of antigen or a standard curve based thereon can be added to this measurement kit. This makes it possible to easily measure an unknown concentration of antigen in a sample. In addition, the kit can add an enzyme substrate when the reporter molecule is an enzyme, and a washing solution as necessary.
[0020]
Next, the second method of the present invention will be described. In this second measurement method, VH labeled with a first reporter molecule and VL labeled with a second reporter molecule are mixed with an antigen-containing sample, and the amount of change in the interaction between the first reporter molecule and the second reporter molecule is determined. This is a method of measuring the amount of Fv formation by both fragments as an index. In this method, it is not necessary to prepare a polypeptide immobilized on a solid phase. Moreover, it is not necessary to wash away the excess labeled polypeptide after Fv formation.
[0021]
As the first reporter molecule and the second reporter molecule, for example, different types of fluorescent dyes having different fluorescence wavelengths and excitation wavelengths (for example, fluorescein and rhodamine X) can be used. That is, when such a combination of fluorescent dyes approaches within a certain distance (about 4 nm), a phenomenon called “fluorescence resonance energy transfer” occurs between the dyes, and one fluorescence is used to excite the other dye, resulting in a result. Will emit longer wavelength fluorescence. Specifically, fluorescein is excited at 490 nm and emits fluorescence at 530 nm. If rhodamine X is present in the vicinity, fluorescence energy at around 530 nm is transferred to rhodamine X, and fluorescence having a longer wavelength (603 nm) is emitted. It comes out. The distance between the amino terminus of each VH chain and VL chain of antibody molecules known to date by X-ray structural analysis is about 3.5 nm, and the distance at which fluorescence resonance energy transfer can be sufficiently detected It is.
[0022]
Therefore, the amino terminus of each of VH and VL can be labeled with a different type of fluorescent dye, and the amount of VH-VL complex can be measured using the change in fluorescence wavelength as an index. As described above, since the amount of VH-VL complex depends on the antigen concentration, the antigen concentration can be accurately measured by the detection.
This second measurement method can be carried out with a set of VH labeled with a first reporter molecule and VL labeled with a second reporter molecule, such set comprising a first reagent containing labeled VH. And a second reagent containing labeled VL. Such a kit may comprise a standard solution or a standard curve.
[0023]
【Example】
Reference examples and examples will be described below, and the embodiments of the present invention will be described in more detail.
Reference example
Using the filamentous phage as a reporter molecule, the concentration of chicken egg white lysozyme (HEL) in the sample was measured.
(A) Preparation of VL region polypeptide of antibody HyHEL-10 against HEL
VH and VL chain structural genes of the antibody HyHEL-10 and the vector plasmid pKTN2 encoding the pelB signal peptide sequence upstream of each (Tsumoto, K. et al., J. Biol. Chem. 269, 28777-28782, 1994) ), A portion 670 bp encoding pelB, VL and ssi transcription termination sequences was cleaved with restriction enzymes Nhel and EcoRI and purified by agarose gel electrophoresis. This DNA fragment was ligated with a DNA fragment obtained by cleaving a vector pET20b (Novagen Inc.) having a T7 promoter with restriction enzymes XbaI and EcoRI to prepare a VL expression vector pETVLhel. E. coli BL21 (DE3) having T7 polymerase on the genome was transformed with this plasmid, and cultured in LB medium at 30 ° C. The cells that reached the saturation cell concentration in 1 l medium were collected by centrifugation, suspended in fresh LB medium containing 0.5 mM IPTG, and then cultured for another 24 hours. After the cells were collected by centrifugation, ammonium sulfate was added to the supernatant until 66% saturation, and the protein in the supernatant was precipitated. The precipitate collected by centrifugation was dissolved in a small amount of water, dialyzed against 10 mM phosphate buffer (pH 7.0), and then DEAE-cellulose was added to ¼ volume to adsorb proteins other than VL in the medium. By repeating this treatment twice, a VL polypeptide having a purity of 95% or more was obtained.
(B) Preparation of M13 phage displaying VH region polypeptide of antibody HyHEL-10 against HEL
Downstream of the pelB signal peptide sequence, HyHEL-10 VH structural gene and part of the VL structural gene, c-myc tag, M13 phage gene3 protein (g3p) C-terminal part, M13 replication origin, ampicillin resistance gene, and pUC In order to remove the extra VL structural gene part from the plasmid pluck2001 (plasmid in which the vector part of pluck2000 (Japanese Patent Application No. 7-129516) is pTZ19U instead of pTZ18U) having a plasmid-derived replication origin, the following operation is performed. went. In order to combine the g3p signal sequence and the VH structural gene with the downstream c-myc tag and the g3pC terminal sequence in accordance with the codon reading frame, two nucleotide sequences respectively shown in SEQ ID NOs: 1 and 2 in the sequence listing Using 50 pmol of each primer, PCR reaction was performed in 100 μl of a reaction solution consisting of 2.5 units of Pfu DNA polymerase (Stratagene), final concentration of 0.2 mM dNTPs, 10 μl of 10 times concentration reaction solution using 1 ng of pluck2001 as a template. . The resulting 380 bp DNA fragment was digested with restriction enzymes SfiI and NotI and ligated with the vector fragment of pluck2001 that was also digested with SfiI and NotI. It was confirmed that the target plasmid was produced by restriction enzyme digestion, and this was named pluck2010. E. coli XL-1 Blue (Δ (lac), endA1, gyrA96, hsdR17 (rk ^- , Mk ⁺ ), RecA1, relA1, supE44, thi1, [F ′, lacIq, lacZΔM15, proAB, Tn10 (tet ^r )]) Was transformed with pluck2010, and the resulting colonies were cultured overnight at 37 ° C. in 5 ml of LB medium containing 12.5 mg / l tetracycline and 1% glucose. Of these, 50 μl was replaced with 25 μl of M13 VCS phage (StratageneSC20000251,> 10 ¹¹ pfu / ml), left at 37 ° C. for 20 minutes, transferred to LB medium containing 5 ml of 100 mg / l ampicillin and 70 mg / l kanamycin, and cultured with shaking at 37 ° C. for 16 hours. The culture solution was centrifuged to remove the cells, and the supernatant was stored at 4 ° C. until used as a phage solution.
(C) Immobilization of VL on microtiter plate and ELISA using VH-phage
In order to immobilize VL or HEL on the microtiter plate, first, each was biotinylated using biotin-NHS (Pierce) according to the attached manual. On the other hand, streptavidin (Wako) dissolved in PBS at 10 mg / l was poured into a microtiter plate (Falcon 3912) at 100 μl per well and adsorbed overnight at 4 ° C., after which it was removed and 200 μl of binding buffer (2% Blocking was performed with skim milk / PBS) at room temperature for 1 hour. The streptavidin plate thus obtained was washed twice with PBS containing 0.1% Tween 20 (PBS-T), and then biotinylated lysozyme was diluted with PBS to 10 mg / l, and 100 μl per well was poured at room temperature. Every other hour, and this was removed and washed twice with PBS-T. To this, 10 μl of a sample containing 0, 1, 10 mg / l VL in PBS and 90 μl of a VH-phage solution previously mixed with an equal amount of binding buffer 30 minutes before were added at 37 ° C. Incubated for hours. After washing twice, 100 μl of 1/5000 diluted peroxidase-labeled anti-M13 antibody (Pharmacia) was added and washed 5 times at 37 ° C. for 1 hour, followed by coloration using ortho-phenylenediamine according to a conventional method. The absorbance at 490 nm was measured by the method to quantify the M13 phage immobilized on the plate. An experiment using biotinylated VL instead of biotinylated lysozyme and lysozyme instead of VL in the sample was performed in the same manner, and the results were compared.
[0024]
The results are as shown in FIG. In FIG. 2, VL1-0 is a batch 1 of biotinylated VL (350 μl 0.1 mol / 1 NaHCO 3). _Three 500 μg purified VL in pH 8.3, 150 mmol / 1 NaCl and 100 μg biotin-NHS in 3.5 μl DMSO were mixed and reacted at room temperature for 30 minutes, then dialyzed against PBS containing 0.02% sodium azide ) Was immobilized on a plate and incubated with VH phage at a concentration of 0 μg / ml HEL. Therefore, for VL1, measurements were performed on samples with HEL concentrations of 0, 1 and 10 μg / ml. Further, the measurement was performed in the same manner when the batch 2 of biotinylated VL (prepared using 1 μl of biotin-NHS solution instead of 3.5 μl of biotin-NHS solution of VL2 and VL1) was immobilized. Further, HEL-0 means that biotinylated HEL was immobilized on a plate and incubated with VH-phage and VL fragment at a concentration of 0 μg / ml. The HEL concentrations are 0, 1 and 10 μg / ml. Moreover, about VH-phage, it measured about three types (phage 1,2,3) produced by culturing from an independent colony, respectively. As is clear from FIG. 2, the binding of M13 phage according to the increase in the concentration of the coexisting protein (HEL or VL) in the sample both when the VL is immobilized on the plate and when the HEL is immobilized. It was confirmed that the amount increased.
(D) Measurement of HEL concentration in sample by ELISA using VH-phage
By the same method as in (c) above, biotinylated VL was immobilized on a plate, and the amount of VH-phage bound was measured by varying the HEL concentration in the sample to prepare a calibration curve. The results are as shown in FIG. 3, and it was confirmed that the antigenic substance having a final concentration of 0.015 μg (15 ng) / ml or more can be measured with high sensitivity and good reproducibility by the method of the present invention. This measurement sensitivity is almost the same as that of the conventional sandwich ELISA method.
Example 1
HEL concentration in the sample was measured using alkaline phosphatase as a reporter molecule.
(A) Construction of Escherichia coli alkaline phosphatase expression plasmid
Chromosomal DNA of Escherichia coli XL1-blue was extracted according to the method of Molecular Cloning 2nd edition (Sambrook, Fritsh, Maniatis, 1989), and a portion 1450 bp encoding alkaline phosphatase (gene name: PhoA, EC3.1.3.1.) Was extracted. Amplified by PCR. At that time, in order to add restriction enzyme NotI sites to both ends of the PCR product, oligonucleotides whose base sequences were shown in SEQ ID NOs: 3 and 4 in the sequence listing were used as PCR primers. Specifically, each primer is 50 pmol and 2.5 units each. Taq In 100 μl reaction solution to which DNA polymerase was added, 35 cycles of PCR reaction were performed using 1 ng of chromosomal DNA of E. coli XL1-blue as a template. The obtained alkaline phosphatase gene fragment was cut with the restriction enzyme NotI, cut out by agarose electrophoresis, and purified. On the other hand, E. coli expression vector pET20b (Novagen Inc.) having T7 promoter, polyhistidine sequence and the like was cleaved with restriction enzyme NotI, treated with bovine intestinal phosphatase, and then ligated with the above alkaline phosphatase gene fragment. The target plasmid was confirmed by restriction enzyme digestion and named pAP.
(B) Preparation of VH-alkaline phosphatase expression plasmid
VH and VL structural genes of the antibody HyHEL-10, and a vector plasmid pKTN2 (Tsumoto. K. et al., J. Biol. Chem., 269, p28777-28782, 1994) encoding the pelB signal peptide sequence upstream of each. ), A portion 480 bp encoding pelB and VH was amplified by the PCR method to obtain a target DNA fragment. At that time, in order to add a T7 promoter sequence at the 5 ′ end of the PCR product and a restriction enzyme HindIII cleavage site at the 3 ′ end, oligonucleotides whose nucleotide sequences are shown in SEQ ID NOs: 5 and 6 in the sequence listing were used as PCR primers. Using. Specifically, each primer is 50 pmol and 2.5 units each. Pfu PCR reaction was performed using pKTN as a template in a 100 μl reaction solution to which DNA polymerase was added. The obtained fragment was cleaved with restriction enzymes EcoRV and HindIII, purified by agarose electrophoresis, and then ligated with the above-described pAP cleaved with restriction enzymes EcoRV and HindIII and purified by agarose electrophoresis. A phosphatase (VH-AP) expression plasmid was prepared. The plasmid was confirmed by restriction enzyme digestion and named pVHAP.
(C) Expression and purification of VH-AP chimeric protein in E. coli
Escherichia coli BL21 (DE3) LysS, which has T7 polymerase on its genome, was transformed with pVHAP by the calcium chloride method and used in LB medium containing 1.5% agar containing antibiotics (50 mg / l ampicillin, 34 mg / l chloramphenicol). The cells were cultured overnight at 37 ° C. to form colonies. The colonies were transferred to 5 ml of LB medium supplemented with antibiotics (50 mg / l ampicillin, 34 mg / l chloramphenicol) and further cultured at 28 ° C. overnight. The cells reaching the saturated cell concentration were collected by centrifugation, suspended in 50 ml of fresh LB medium containing antibiotics (50 mg / l carbenicillin, 34 mg / l chloramphenicol), and further 3-4 at 28 ° C. Cultured for hours and the medium was scaled up to 1 liter. Cell density is OD ₆₀₀ When reaching = 0.3-0.4, VH-AP chimeric protein was expressed by induction with 0.1 ml of IPTG. After culturing overnight and using 20 μl of the culture solution as it was, and confirming the expression of the target protein by SDS-polyacrylamide electrophoresis, the cells were collected by centrifugation, and the culture supernatant and the cells were separated. To the supernatant, ammonium sulfate was added until the saturation reached 65%, and the protein in the supernatant was precipitated. The precipitate collected by centrifugation was added to the sonication buffer (50 mM NaH ₂ PO _Four , 10 mM Tris-HCl: pH 8.0). The cells were suspended in a sonication buffer containing 1 mM DTT, the cell membrane was disrupted using a French press, and the insoluble fraction was removed by ultracentrifugation (32000 rpm, 1 hour) to prepare a lysate.
[0025]
For purification, TALON using the 6 × His sequence encoded at the C-terminus of the VH-AP chimeric protein is used. ^TM The purification was performed in two stages: purification using a metal chelating column and purification using an anion exchange column. First, TALON ^TM Metal Chelating Resin (Clontech Laboratories Inc., 2 ml of column head) and 20 ml of protein solution were gently stirred at 4 ° C. for 1 hour in a 50 ml tube to adsorb the protein having the polyhistidine sequence, transferred to a 5 ml column, and 20 mM MES. Elute with -Na buffer. Of the obtained fractions, those containing protein are collected, put into a dialysis tube, concentrated by sprinkling an appropriate amount of PEG6000 from the outside, dialyzed into a 20 mM Tris-Cl solution (pH 8.0), and then an anion. It was applied to an exchange column (MonoQ HR5 / 5, Pharmacia Biotech Co.). The obtained fraction was confirmed by SDS polyacrylamide electrophoresis, and only the fraction containing only the target VH-AP chimera protein was recovered. After concentration, Tris solution (0.1M Tris-Cl, 0.1M Dialyzed to NaCl, pH 8.0). Through this series of operations, the chimeric protein could be purified from the culture supernatant and cell lysate until it was identified as a single band in SDS polyacrylamide electrophoresis. The obtained protein was measured for concentration using the BCA protein assay (Pierce) and then stored at 4 ° C. until used for measurement.
(D) Measurement of HEL using VH-alkaline phosphatase
Using a VL plate prepared in the same manner as in the Reference Example, 10 μl of a sample containing HEL at each concentration in PBS and 90 μl of the above VH-AP chimeric protein solution (4 μg / ml in 0.1 M Tris HCl pH 8.0) In addition to the wells, they were incubated for 2 hours at room temperature. Each well was washed 4 times with PBS containing 0.1% Tween (PBS-T), 100 μl of substrate (1 mM 4-nitrophenylphosophate, Wako) was added per well, and coloration (yellow) after reaction for 30 minutes and 1 hour ) Was measured at an absorbance of 410 nm.
[0026]
The results are as shown in FIG. This FIG. 4 is a plot of the absorbance of the reaction 1 hour book against the final concentration of HEL, and the plotted value is the average of the two measurements. As is clear from FIG. 4, it was confirmed that an antigenic substance having a final concentration of 1 ng / ml or more can be measured with high sensitivity and good reproducibility by the method of the present invention. In addition, the measurement sensitivity when this alkaline phosphatase is used as a reporter molecule is about 10 times that in Example 1 using a phage on fiber.
Example 2
The association of fluoresceinated VH and rhodamine X VL was detected by fluorescence energy transfer.
(A) Preparation of VH and VL
VH and VL structural genes of antibody Hy-HEL-10 against HEL and a vector plasmid pKTN2 (Tsumoto, K. et al., J. Bio. Chem., 269, p28777- encoding the pelB signal peptide sequence upstream of each) 28782, 1994), and the Fv fragment of HyHEL-10 was prepared according to a known method. That is, Escherichia coli BL21 (DE3) was transformed with pKTN2, cultured at 30 ° C. in 5 ml of LB medium containing 50 mg / l ampicillin, planted with 50 ml and 1 l in the same medium, and then cultured until a saturated cell concentration was reached. After one collection, the cells are cultured in the same medium containing 0.4 mM IPTG for 24 hours, and the centrifuged supernatant is salted out with 66% saturated ammonium sulfate, and the precipitate is diluted with 50 mM phosphoric acid (pH 7.2) and 0.2 M NaCl. Dialyzed. After centrifuging (10 krpm, 10 min.), Equilibrate with HEL affinity column in the same buffer (10 mg / ml HEL immobilized on Pharmacia CNBr activated Sepharose 4B according to the addition manual) The sample was adsorbed, washed with 100 ml of 0.1 M Tris-HCl (pH 8.5) and 0.5 M NaCl, and then eluted with 10 ml of 0.1 M glycine buffer (pH 2.0). Immediately after elution, the solution was neutralized with an equal volume of 1M Tris-HCl (pH 7.5).
[0027]
About 10 mg of the obtained Fv fragment was dialyzed against 50 mM Tris-HCl (pH 8.8) and then separated into VH and VL by an anion exchange chromatography column (MonoQ HR5 / 5, Pharmacia Co.). That is, since VL hardly binds to the column under these chromatographic conditions, it was possible to relatively easily separate the unbound fraction from VL and the bound fraction from VH. When the purity of each polypeptide after separation was confirmed by SDS polyacrylamide electrophoresis, it was 90% or more.
(B) VH and VL fluorescent dye labels
Purified VH (0.5 ml) was dialyzed against 0.2 M sodium phosphate (pH 7.0) and 0.1 M NaCl, and the dialyzate was subjected to 10 mM (about 5 mg / ml) fluorescein succinimide ester (Molecular Probes Inc.) in dimethyl sulfoxide. ., Eugene, USA) was added in an amount of 4.5 μl, mixed well, and allowed to react at 4 ° C. for 10 hours. At the same time, 4 μl of 10 mM (about 6 mg / ml) rhodamine X succinimide ester (Molecular Probes Inc., Eugene, USA) in dimethyl sulfoxide was added to 235 μl of purified VL (0.5 ml) dialyzed against the same buffer. The mixture was mixed and reacted at 4 ° C. for 10 hours. The molar ratio of protein: pigment at this time was 1: 2. The reaction was stopped by adding an equal volume of 1M Tris-HCl (pH 7.5), and gel filtration was performed with a PD-10 column (Pharmacia Co.) equilibrated in advance with 0.2M sodium phosphate (pH 7.0) and 0.1M NaCl. To remove unreacted dye.
(C) Measurement of HEL concentration
Add 20 μl and 25 μl of the above fluoresceinated VH and rhodamine X VL to a solution (2 ml) of 0.2 M sodium phosphate (pH 7.0), 0.1 M NaCl and 1% bovine serum albumin, respectively, and place them in a cuvette. The fluorescence intensity was measured at 4 ° C. with an excitation wavelength of 490 nm and a fluorescence wavelength of 500 to 650 nm using a Hitachi spectrofluorometer 850 type. While stirring with a stirrer, HEL was sequentially added from a final concentration of 0.1 μg / ml to 1 mg / ml, and changes in the fluorescence spectrum were observed.
[0028]
As a result, it was observed that as the HEL concentration increased, the fluorescence intensity at 530 nm decreased and the fluorescence intensity at 603 nm derived from energy transfer increased. The time required for this change was within a few seconds. From the results of performing this experiment three times independently, as shown in FIG. 5, it was possible to measure the antigen HEL concentration with good reproducibility by taking the amount of change in the ratio of the fluorescence intensities of the two.
[0029]
【The invention's effect】
As described above in detail, according to the present invention, the antigen concentration in a sample can be easily and rapidly performed with the same measurement sensitivity as that of the conventional sandwich ELISA method.
[0030]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic view illustrating a measurement procedure according to the present invention.
FIG. 2 is a bar graph showing the amount of VH phage bound to biotinylated VL or chicken egg white lysozyme immobilized on a plate.
FIG. 3 is a calibration curve of chicken egg white lysozyme concentration prepared based on the binding amount of VH-phage to biotinylated VL immobilized on a plate.
FIG. 4 is a graph plotting the absorbance indicating the amount of alkaline phosphatase-labeled VH bound to biotinylated VL immobilized on a plate for each hen egg white lysozyme concentration.
FIG. 5 is a graph plotting the fluorescence ratio of fluoresceinated VH and rhodamine X-modified VL for each hen egg white lysozyme concentration.

Claims (8)

(A) preparing a VH region polypeptide and VL-region polypeptide of an antibody specific to the antigen;
(B) a step of labeling one polypeptide with a reporter molecule to form a labeled polypeptide, and immobilizing the other polypeptide to a solid phase to form an immobilized polypeptide ;
Step (c) Ru brought into contact with the solid phase antigen-containing sample and the labeled polypeptide; and
Step (d) measuring the reporter component child of bound labeled polypeptide to the immobilized polypeptide
A method for measuring an antigen concentration in a sample, comprising: The polypeptide to be immobilized on the solid phase is pre-biotinylated or modified with a tag sequence and the solid phase absorbs or chemically binds avidin or streptavidin, thereby biotin-avidin binding, biotin-streptavidin binding or tag The method of claim 1, wherein the polypeptide is immobilized on the solid phase via a sequence-streptavidin linkage. (A) preparing a VH region polypeptide and VL-region polypeptide of an antibody specific to the antigen;
(B) labeling the VH region polypeptide with a first reporter molecule and labeling the VL region polypeptide with a second reporter molecule ;
(C) mixing the antigen-containing sample with the labeled VH region polypeptide and the labeled VL region polypeptide ; and
(D) a step of measuring a change index value of the first reporter molecule or the second reporter molecule induced by the interaction between the first reporter molecule and the second reporter molecule
A method for measuring an antigen concentration in a sample, comprising: A labeled polypeptide labeled with a reporter molecule, which is a VH region polypeptide or VL region polypeptide of an antibody specific for an antigen, and which is a filamentous phage fused to the polypeptide. The labeled polypeptide according to claim 4, wherein the filamentous phage is previously labeled with an enzyme or a fluorescent dye. A labeled VH-region polypeptide and the VL region is labeled with a second reporter molecule polypeptide de or Ranaru a set of labeled polypeptide in the first reporter molecule, VH-region polypeptide and the VL-region polypeptide antigen A set of labeled polypeptides derived from a specific antibody. A solid phase having an immobilized VL region or VH region polypeptide; and
Reagent comprising labeled VH region or VL region polypeptide
A kit for measuring an antigen concentration in a sample, comprising: A first reagent containing a labeled VH region polypeptide ; and a second reagent containing a labeled VL region polypeptide
Comprising a kit for measuring the concentration of antigen in the sample.

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