JP4413371B2 - Method for measuring PSA and its reagent - Google Patents

Description

なお、ＰＳＡ測定キットは「タンデム−Ｒ」（ハブリテック社製）を用いた。タンデム−Ｒは免疫放射定量法（ＩＲＭＡ）によりＰＳＡの濃度を測定するものである。
【００３１】
表１の結果から実施例ではいずれも標準的なＰＳＡ測定キットの「タンデム−Ｒ」の測定値とよい一致を示し、比較例ではサンプルＡ、Ｂ、Ｃのいずれも標準的なＰＳＡ測定キットの「タンデム−Ｒ」と乖離した測定値を示し、本発明の有効性が証明された。
また、図１（比較例）では検量曲線がｆＰＳＡとＡＣＴ−ＰＳＡで大きくずれているため、トータルのＰＳＡの測定値は、被検液中のｆＰＳＡとＡＣＴ−ＰＳＡの存在比により大きく変化することが分かる。一方、図２〜４（実施例）ではｆＰＳＡとＡＣＴ−ＰＳＡの反応性が一致しているため、ｆＰＳＡを標準物質として用いた検量曲線の測定値がトータルＰＳＡの測定値となる。
従って、本発明によれば被検液中のｆＰＳＡとＡＣＴ−ＰＳＡの反応性が等しくなるため、ｆＰＳＡを標準物質とした検量曲線を用いて被検液中のｆＰＳＡとＡＣＴ−ＰＳＡの存在比が異なっていても一定の総ＰＳＡの定量値が得られる。この結果、前立腺癌の信頼性の高いスクリーニング、診断ができるようになった。
【００３２】
【発明の効果】
本発明により、ｆＰＳＡとＡＣＴ−ＰＳＡの反応性を等しくして、試料中の総ＰＳＡを迅速・簡便に測定でき、また、大量の被検液を短時間で測定でき、得られた測定値は本発明の原理からｆＰＳＡもＡＣＴ−ＰＳＡも同じ濃度で測定することができるため、ｆＰＳＡとＡＣＴ−ＰＳＡの比の異なる被検液でも同じ定量値が得られ、前立腺癌のスクリーニングを効率良く進めることが可能となった。
【図面の簡単な説明】
【図１】図１は比較例の、ｆＰＳＡとＡＣＴ−ＰＳＡの検量曲線を示す
【図２】図２は実施例１の、ｆＰＳＡとＡＣＴ−ＰＳＡの検量曲線を示す
【図３】図３は実施例２の、ｆＰＳＡとＡＣＴ−ＰＳＡの検量曲線を示す
【図４】図４は実施例３の、ｆＰＳＡとＡＣＴ−ＰＳＡの検量曲線を示す[0001]
[Industrial application fields]
The present invention relates to a method for measuring prostate-specific antigen (hereinafter abbreviated as PSA) by immunoagglutination and its reagent.
[0002]
[Prior art]
Prostate cancer is a malignant disease seen in men, and is the most common solid tumor in the United States. Even in Japan, the rate of increase is remarkable, and it is estimated that it will increase by 3.9 times in 1995, which is the highest among all tumors. Prostate cancer is a slow-growing tumor and has features that anti-androgen therapy is likely to be effective, and early detection is important because if it is detected early without metastasis or invasion, the opportunity to cure it is important It is.
Until now, prostate cancer has been diagnosed by rectal palpation and transrectal echocardiography, and if these tests are suspected to be prostate cancer, a definitive diagnosis has been obtained by needle biopsy. However, both rectal palpation and transrectal echo were enteral operations, which accompanied the patient's shame, and delaying the visit to the urologist contributed to the delay in early detection.
PSA secreted from prostate epithelial cells and also produced by prostate cancer cells is a kind of serine protease and is a glycoprotein having a molecular weight of 33,000 to 34,000 daltons. The amino acid sequence has also been reported (Proc. Natl. Acad. Sci. USA, 83, 3166 (1986)). PSA exists in blood as a bound form with a protease inhibitor and an unbound free prostate cancer specific antigen (abbreviated as fPSA). Most of the PSA in the blood is conjugated, α1-antichymotrypsin complex prostate specific antigen (hereinafter abbreviated as ACT-PSA) that is bound to α1-antichymotrypsin and α2 macroglobulin that is bound to α2 macroglobulin. There are complex prostate specific antigens and the like. Among these, the α2 macroglobulin complex prostate specific antigen cannot be measured immunologically because the whole PSA molecule is covered with α2 macroglobulin and the antigenic activity is lost. That is, there are two types of PSA that can be measured by the immunization method, fPSA and ACT-PSA.
PSA is also positive in early prostate cancer cases in serum, and prostate cancer may be detected even when there are no findings in rectal palpation or transrectal echo, and its performance as an index for screening prostate cancer patients is It has been found to be superior to rectal palpation and transrectal echo.
[0003]
[Problems to be solved by the invention]
However, in the kit for measuring the total amount of PSA in serum over 20 kinds marketed in Japan, there is a problem that the degree of deviation between the measured value and the true value varies depending on the test solution. This main cause is considered to be due to the difference in the reactivity of the measurement reagent for fPSA and ACT-PSA in each kit. PSA is generally known for five types of epitopes, and monoclonal antibodies are obtained for each. In radioimmunoassay and enzyme immunoassay in which reaction with antigen is carried out in two steps, attempts have been made to equalize the reactivity of fPSA and ACT-PSA by combining these monoclonal antibodies. Optimal monoclonal antibodies are commercially available. However, in the measurement method based on the agglutination reaction such as the latex method, the antigen-antibody reaction is in one stage, so it is difficult to equalize the reactivity of fPSA and ACT-PSA.
[0004]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found a total PSA measurement method by an aggregation method that can be applied to a general-purpose automatic analyzer, and have completed the present invention.
[0005]
That is, the present invention
(1) A measurement method characterized by adding an antibody against fPSA that does not react with ACT-PSA to a test solution and then adding a carrier sensitized with the antibody against PSA in the measurement by the aggregation method of PSA.
(2) The method according to (1) above, wherein the antibody against fPSA that does not react with ACT-PSA is a monoclonal antibody or an antigen-binding fragment thereof.
(3) The measurement method according to (1) or (2) above, wherein the carrier sensitized with an antibody against PSA is latex.
[0006]
(4) The method according to any one of (1) to (3) above, wherein the antibody against PSA sensitized to the carrier is a polyclonal antibody, two or more kinds of monoclonal antibodies, or a combination thereof.
(5) A reagent for measuring PSA comprising a reagent containing an antibody against fPSA that does not react with ACT-PSA and a carrier sensitized with an antibody against PSA.
(6) The measuring reagent according to (5), wherein the carrier sensitized with an antibody against PSA is latex.
About.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The PSA agglutination method of the present invention is based on the addition of an antibody against fPSA that does not react with ACT-PSA to the test solution, and then a carrier sensitized with the antibody against PSA.
In the present invention, the region capable of binding to α1-antichymotrypsin of fPSA is bound to a somewhat large molecular weight protein, specifically an antibody, thereby bringing the molecular weights of fPSA and ACT-PSA close to each other and aligning the number of epitopes. The reactivity of fPSA to the antibody against PSA and the reactivity of ACT-PSA were brought close to each other, making it possible to measure the carrier sensitized with the antibody against PSA and both in an equimolar correspondence.
The antibody against fPSA used in the present invention is an antibody that does not react with ACT-PSA and that recognizes a site where α1-antichymotrypsin and PSA bind. This antibody may be obtained by removing an antibody that reacts with ACT-PSA from a polyclonal antibody obtained by immunizing an animal with PSA, or may be a commercially available antibody, preferably a site where α1-antichymotrypsin and PSA bind to each other. Monoclonal antibody that recognizes. The antibody may be the whole antibody, but may be an F (ab ′) ₂ fragment or Fab fragment that is an antigen-binding fragment obtained by cleaving the antibody with an enzyme such as pepsin or papain. A Fab ′ fragment obtained by cleaving the -SS- bond of the F (ab ′) ₂ fragment and stabilizing it may also be used. The concentration of the antibody used may be 0.05 to 10 μg / mL in the solution at the time of reaction with fPSA, but a concentration of 0.25 to 2.5 μg / mL is preferable and is present in excess of fPSA in the test solution. It is necessary to.
The test solution used in the present invention is not particularly limited as long as it is a sample containing PSA, but preferably includes blood, plasma, serum and the like, and serum is particularly preferable.
[0008]
The antibody against PSA used by sensitizing the carrier in the present invention is an antibody that reacts with both ACT-PSA and fPSA, and a polyclonal antibody obtained by immunizing an animal usually used for obtaining the antibody is this antibody. It is effective for the aggregation reaction used in the invention. The animal species used for immunization may be commonly used rabbits, sheep, goats and the like. Further, a combination of two or more anti-PSA monoclonal antibodies that react with ACT-PSA, or a combination of a polyclonal antibody and a monoclonal antibody may be used. The whole antibody may be used, but it may be an F (ab ′) ₂ fragment, Fab fragment, or thiol group-stabilized Fab ′ fragment cleaved with the above enzyme according to a conventional method.
[0009]
The carrier for sensitizing the antibody against PSA used in the present invention is not particularly limited, but latex is preferred. As the latex, a general latex made of polystyrene used in the physical adsorption method used in immunological measurement methods can be used, and the material is not particularly limited as long as it is suitable for the sensitization method. The particle size is preferably 50 to 1000 nm, more preferably 400 to 700 nm, which enables highly sensitive measurement. Also, it is possible to use not only a monodisperse latex having a uniform particle size but also a latex having a wide distribution, or a latex in which different particle sizes are mixed.
[0010]
As a method for sensitizing an antibody to latex, a generally used physical adsorption method can be adopted, but a chemical bonding method can also be adopted. To illustrate physical adsorption, polystyrene latex with a particle size of 450 nm is dispersed in a suitable buffer solution at a concentration of 1%, and an antibody cleaved with pepsin is dissolved in the same buffer solution prepared to a concentration of 1 mg / mL. The same amount was mixed and sensitized in the refrigerator overnight, and the same amount of 1% bovine serum albumin (BSA) solution was further added, and the mixture was further left overnight in the refrigerator with gentle shaking. The supernatant can be exchanged with a 1% BSA-containing buffer using a centrifuge to obtain a sensitized latex.
The sensitized latex is prepared to a concentration of about 0.05 to 0.2% and used for the reaction.
[0011]
When measuring PSA by this antigen-antibody reaction, it is preferable to improve stability by adding an immunologically inactive protein such as BSA. Furthermore, the addition of a water-soluble polymer that promotes the aggregation reaction is preferable. Specific examples include polyethylene glycol, dextran, polyvinyl pyrrolidone, polyvinyl acetamide, polyvinyl formamide, and the addition amount varies depending on the molecular weight, but is about 0.2% to 2.0% in the measurement solution. An amount indicating is used.
[0012]
The antigen-antibody reaction in the present invention is performed in a buffer solution. When sensitizing the carrier and agglutination reaction, the optimum buffer type, concentration, and pH are selected for the buffer solution, respectively, and commonly used phosphate buffer solution, Tris-HCl buffer solution, carbonate buffer solution, glycine buffer solution. Liquid, Good's buffer, etc. can be used as appropriate. The concentration of the buffer in the measurement solution is about 10 mM to 500 mM. Moreover, the pH is often used in the neutral to basic range, and is usually in the range of 7.0 to 9.5. For example, a 1% BSA-containing buffer is often used in the pH range of 7.2 to 8.5.
[0013]
For example, the PSA measurement method of the present invention is performed as follows. The fPSA in the test solution is reacted with an anti-fPSA monoclonal antibody that does not react with ACT-PSA as a first reaction to form antibody-bound fPSA, and ACT-PSA remains as it is. As a second reaction, antibody-bound fPSA and ACT-PSA are allowed to undergo an antigen-antibody reaction with an antibody-sensitized latex against PSA to aggregate latex particles. The degree of aggregation is measured, and the PSA concentration in the test solution is quantified by comparing with the degree of aggregation of a standard solution whose PSA concentration is known.
[0014]
At this time, there are various methods for measuring the degree of aggregation, but a method using a general-purpose biochemical analyzer is convenient. For example, the first reaction is 5 minutes, the second reaction is also 5 minutes, the absorbance is measured 30-50 times during this time, the change in absorbance is detected, and the concentration of the test solution is determined from the calibration curve of the standard solution. calculate. In the latex agglutination method, absorbance at a wavelength of 500 to 900 nm is usually used, and the amount of change in absorbance in the second reaction is generally used for quantification, and the method of the present invention can also be measured by this method.
[0015]
The PSA measurement reagent of the present invention is characterized by comprising an antibody against fPSA that does not react with ACT-PSA and a carrier sensitized with an antibody against PSA. The above-mentioned antibodies can be used for the antibodies against PSA used in the present invention. The carrier sensitized with an antibody against PSA is preferably latex, and specifically, those described above are used.
[0016]
【Example】
EXAMPLES Hereinafter, although a comparative example and an Example demonstrate this invention concretely, this invention is not limited to these Examples.
[0017]
Comparative Example Measurement of PSA Using Anti-PSA Goat Antibody Reagents having the following compositions were prepared, and 0, 6.25, 12.5, 25.0, 50.0 and 100.0 ng / mL of fPSA and ACT-PSA The results are shown in FIG. 1, and three samples of PSA-containing human serum were measured. The results are shown in Table 1. The concentration of human serum was determined using fPSA as a calibration curve. The measurement was performed using an automatic analyzer 7150 (manufactured by Hitachi, Ltd.) with the following parameters.
Analysis method 2 point end measurement point 27-50
Sample volume 20μL
First reagent 200 μL
Second reagent 40μL
Temperature 37 ° C
Measurement wavelength (one wavelength) 750nm
[0018]
Reagent composition first reagent Tris buffer (pH 7.5) 500 mM
BSA 0.5%
NaCl 500 mM
Polyvinylpyrrolidone K-90 0.5%
[0019]
Second reagent Prepared as follows.
1 mL of latex (10% (W / V)) having an average particle size of 480 nm was dispersed in 9 mL of 5 mM glycine buffer (PH9.5), and 1 mL of anti-PSA goat antibody (IgG fraction, 10 mg / mL) (manufactured by BiosPacific) And gently shaken at room temperature for 1 hour. Thereafter, 10 mL of 5 mM glycine buffer (PH9.5) containing 1% (W / V) bovine serum albumin (BSA) was added and shaken at room temperature for 1 hour, and the latex and supernatant were separated by centrifugation, The supernatant was discarded, and latex particles were dispersed in 50 mL of 10 mM Tris buffer (PH7.5) containing 1% (W / V) BSA to obtain a second reagent.
[0020]
Using a measurement automatic analyzer 7150 type, 20 μL of sample is placed in a reaction cell at 37 ° C., and 200 μL of the first reagent is immediately added and mixed. After 5 minutes, the second reagent 40μ is added and mixed with stirring. The 7150 analyzer measures the absorbance at a wavelength of 750 nm every 12 seconds for 10 minutes from the start of the first reaction to the end of the second reaction. The amount of increase in absorbance from 27 to 50 points after the start of the second reaction is measured, and the concentration is calculated by comparison with the amount of increase in absorbance of the standard solution.
[0021]
Example 1 Measurement of PSA Using Anti-fPSA Mouse Antibody (IgG Fraction) and Anti-PSA Goat Antibody A reagent having the following composition was prepared, and 0, 6.25, 12.5, 25.0, 50.0 and 100.0 ng / mL fPSA and ACT-PSA were measured, and the results are shown in FIG. 2. Three PSA-containing human serum samples were measured, and the results are shown in Table 1. The concentration of human serum was determined using fPSA as a calibration curve. For the measurement, an automatic analyzer 7150 type (manufactured by Hitachi, Ltd.) was used, and the same parameters as in the comparative example were used.
[0022]
Reagent composition first reagent Tris buffer (pH 7.5) 500 mM
BSA 0.5%
NaCl 500 mM
Polyvinylpyrrolidone K-90 0.5%
Anti-fPSA specific mouse monoclonal antibody (IgG fraction) (manufactured by BiosPacific) 0.2 μg / mL
[0023]
Second reagent Prepared as follows.
1 mL of latex (10% (W / V)) having an average particle size of 480 nm was dispersed in 9 mL of 5 mM glycine buffer (PH9.5), and 1 mL of anti-PSA goat antibody (IgG fraction, 10 mg / mL) was added thereto at room temperature. Shake gently for hours. Thereafter, 10 mL of 5 mM glycine buffer (PH9.5) containing 1% (W / V) bovine serum albumin (BSA) was added and shaken at room temperature for 1 hour, and the latex and supernatant were separated by centrifugation, The supernatant was discarded, and latex particles were dispersed in 50 mL of 10 mM Tris buffer (PH7.5) containing 1% (W / V) BSA to obtain a second reagent.
Measurement Using the first reagent and the second reagent, measurement was performed in the same manner as in the comparative example.
[0024]
Example 2 Measurement of PSA Using Anti-fPSA Mouse Antibody (F (ab ′) ₂ Fraction) and Anti-PSA Goat Antibody Reagents having the following composition were prepared and 0, 6.25, 12.5, 25.0 50.0 and 100.0 ng / mL of fPSA and ACT-PSA were measured. The results are shown in FIG. 3, and three PSA-containing human sera were measured. The results are shown in Table 1. The concentration of human serum was determined using fPSA as a calibration curve. For the measurement, an automatic analyzer 7150 type (manufactured by Hitachi, Ltd.) was used, and the same parameters as in the comparative example were used.
[0025]
Reagent composition first reagent Tris buffer (pH 7.5) 500 mM
BSA 0.5%
NaCl 500 mM
Polyvinylpyrrolidone K-90 0.5%
Anti-fPSA specific mouse monoclonal antibody (F (ab ′) ₂ fraction) 2.0 μg / mL
[0026]
Second reagent Prepared as follows.
1 mL of latex (10% (W / V)) having an average particle size of 480 nm was dispersed in 9 mL of 5 mM glycine buffer (PH9.5), and 1 mL of anti-PSA goat antibody (IgG fraction, 10 mg / mL) was added thereto at room temperature. Shake gently for hours. Thereafter, 10 mL of 5 mM glycine buffer (PH9.5) containing 1% (W / V) bovine serum albumin (BSA) was added and shaken at room temperature for 1 hour, and the latex and supernatant were separated by centrifugation, The supernatant was discarded, and latex particles were dispersed in 50 mL of 10 mM Tris buffer (PH7.5) containing 1% (W / V) BSA to obtain a second reagent.
Measurement Using the first reagent and the second reagent, measurement was performed in the same manner as in the comparative example.
[0027]
Example 3 Measurement of PSA Using Anti-fPSA Mouse Antibody (Fab Fraction) and Anti-PSA Goat Antibody A reagent having the following composition was prepared, and 0, 6.25, 12.5, 25.0, 50.0 and 100.0 ng / mL of fPSA and ACT-PSA were measured, and the results are shown in FIG. 4. Three PSA-containing human serum samples were measured, and the results are shown in Table 1. The concentration of human serum was determined using fPSA as a calibration curve. For the measurement, an automatic analyzer 7150 type (manufactured by Hitachi, Ltd.) was used, and the same parameters as in the comparative example were used.
[0028]
Reagent composition first reagent Tris buffer (pH 7.5) 500 mM
BSA 0.5%
NaCl 500 mM
Polyvinylpyrrolidone K-90 0.5%
Anti-fPSA specific mouse monoclonal antibody (Fab fraction) 1.2 μg / mL
[0029]
Second reagent Prepared as follows.
1 mL of latex (10% (W / V)) having an average particle size of 480 nm was dispersed in 9 mL of 5 mM glycine buffer (PH9.5), and 1 mL of anti-PSA goat antibody (IgG fraction, 10 mg / mL) was added thereto at room temperature. Shake gently for hours. Thereafter, 10 mL of 5 mM glycine buffer (PH9.5) containing 1% (W / V) bovine serum albumin (BSA) was added and shaken at room temperature for 1 hour, and the latex and supernatant were separated by centrifugation, The supernatant was discarded, and latex particles were dispersed in 50 mL of 10 mM Tris buffer (PH7.5) containing 1% (W / V) BSA to obtain a second reagent.
Measurement Using the first reagent and the second reagent, measurement was performed in the same manner as in the comparative example.
[0030]
Measurement results of human serum containing PSA

As the PSA measurement kit, “tandem-R” (manufactured by Habritech) was used. Tandem-R measures the concentration of PSA by immunoradiometric assay (IRMA).
[0031]
From the results of Table 1, all the examples show good agreement with the measured values of “tandem-R” of the standard PSA measurement kit, and in the comparative examples, all of samples A, B, and C are of the standard PSA measurement kit. The measured value deviated from “Tandem-R” was shown, and the effectiveness of the present invention was proved.
Further, in FIG. 1 (comparative example), the calibration curve is greatly deviated between fPSA and ACT-PSA, so that the total measured value of PSA varies greatly depending on the abundance ratio of fPSA and ACT-PSA in the test solution. I understand. On the other hand, in FIGS. 2 to 4 (Examples), since the reactivity of fPSA and ACT-PSA coincides, the measured value of the calibration curve using fPSA as a standard substance becomes the measured value of total PSA.
Therefore, according to the present invention, the reactivity of fPSA and ACT-PSA in the test solution becomes equal, and therefore the abundance ratio of fPSA and ACT-PSA in the test solution is determined using a calibration curve using fPSA as a standard substance. Even if they are different, a constant quantitative value of total PSA is obtained. As a result, reliable screening and diagnosis of prostate cancer have become possible.
[0032]
【The invention's effect】
According to the present invention, the reactivity of fPSA and ACT-PSA can be made equal, the total PSA in the sample can be measured quickly and easily, and a large amount of the test solution can be measured in a short time. Since fPSA and ACT-PSA can be measured at the same concentration based on the principle of the present invention, the same quantitative value can be obtained even in test solutions having different ratios of fPSA and ACT-PSA, and screening for prostate cancer can be carried out efficiently. Became possible.
[Brief description of the drawings]
FIG. 1 shows a calibration curve of fPSA and ACT-PSA in a comparative example. FIG. 2 shows a calibration curve of fPSA and ACT-PSA in Example 1. FIG. Fig. 4 shows calibration curves of fPSA and ACT-PSA in Example 2. Fig. 4 shows calibration curves of fPSA and ACT-PSA in Example 3.

Claims (3)

In measurement of prostate specific antigen by latex agglutination method, an antibody against free prostate specific antigen that does not react with α1-antichymotrypsin-complexed prostate specific antigen is added to the test solution, and then polyclonal antibody or two types against prostate specific antigen A method for measuring prostate-specific antigen, comprising adding latex sensitized with the above monoclonal antibody or an antibody that is a combination thereof . 2. The measuring method according to claim 1, wherein the antibody against the free prostate specific antigen that does not react with the α1-antichymotrypsin complex prostate specific antigen is a monoclonal antibody or an antigen-binding fragment thereof. A reagent containing an antibody against a free prostate-specific antigen that does not react with an α1-antichymotrypsin complex prostate-specific antigen, and a latex sensitized with a polyclonal antibody against the prostate-specific antigen, two or more monoclonal antibodies, or an antibody that is a combination thereof. A measurement reagent for a latex agglutination method of a prostate-specific antigen comprising a reagent containing the reagent.

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