JP2005326150A - Method for measuring prostate specific antigen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new immunological agglutination measuring method which enables the precise measurement of PSA in serum regardless of the presence ratio of free PSA and α1 anti-chymotrypsin combined PSA, and a measuring reagent. <P>SOLUTION: In the immunological agglutination measuring method of serum PSA using insoluble carrier particles of latex or the like, a monoclonal antibody reacted with both of free PSA and α1 anti-chymotrypsin combined PSA and different in reactivity with respect to both of them is utilized. The measuring reagent used in this measuring method is also disclosed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for measuring prostate-specific antigen, and more particularly to a method for measuring prostate-specific antigen by immunological aggregation.

  Prostate-specific antigen (hereinafter abbreviated as “PSA”) is a serine proteolytic enzyme having a molecular weight of about 34,000 produced from prostate epithelial cells discovered by Wang et al. In 1979. It was revealed that γ-seminoprotein was the same substance from the amino acid sequence and the function of the protease. Although PSA is specific to the prostate, it is not specific to cancer and is also produced by normal prostate cells, so it can be increased in prostate diseases other than prostate cancer such as benign prostatic hyperplasia and prostatitis. It has been reported. However, because of its usefulness such as assessment of the effects of various treatments for prostate cancer, evaluation of recurrence / relapse after treatment, and early detection of residual tumor after radical prostatectomy, Instead, it is positioned as a tumor marker specific to the organ called the prostate.

In adult males, PSA is released in serum at a concentration of about 10 ^-6 times that of seminal plasma. In prostate disease, regardless of whether it is malignant or benign, a significantly large amount of PSA is detected in the serum. . And most of them are protease inhibitors such as α1-antichymotrypsin (hereinafter abbreviated as “ACT”) and α2-macroglobulin (hereinafter abbreviated as “α2M”), which have an action to prevent tissue damage caused by proteolytic enzymes. And a part of it is present as a non-complex free PSA (hereinafter abbreviated as “F-PSA”).

  Usually, PSA is measured as total PSA (hereinafter abbreviated as “T-PSA”), which is a combination of ACT composite type PSA (hereinafter abbreviated as “ACT-PSA”) and F-PSA. Immunological measurement methods such as immunoassay (hereinafter abbreviated as “IRMA method”) and enzyme immunoassay (hereinafter abbreviated as “EIA method”) are the mainstream.

  The clinical significance of PSA has been found to be high in the proportion of F-PSA in non-cancer diseases such as benign prostatic hyperplasia while ACT-PSA increases in prostate cancer. Therefore, since it is possible to distinguish prostate cancer from benign prostatic hyperplasia from the measured values of T-PSA, ACT-PSA, and F-PSA in serum, early detection of prostate cancer is possible. Note that PSA bound to α2M is not recognized by anti-PSA antibodies because the entire PSA molecule is covered with α2M, and as a result, it is said that immunological measurement as PSA is not possible. Thus, the immunologically measured complex PSA is usually ACT-PSA.

  By the way, as a big problem of T-PSA measurement in a biological sample, particularly serum, there is a difference between 20 or more kinds of reagent kits commercially available for measuring T-PSA, that is, the measurement value varies greatly depending on the kit. . This is largely due to the difference in reactivity of each reagent kit with respect to F-PSA and ACT-PSA. In particular, it has been reported that the tendency becomes prominent when the ACT-PSA / F-PSA ratio becomes high, but this is caused by a variation in reactivity to ACT-PSA.

  Since 1997, a survey aimed at standardization of serum T-PSA has been conducted by the Japanese Urological Association, and it is less than 20 ng / mL (especially in the gray zone of 4 to 10 ng / mL), which is the most problematic in clinical and screening settings. There is a need for a reagent kit that can be measured properly and that shows not only a purified sample but also a serum sample with similar reactivity. Therefore, in order to accurately measure T-PSA, there is a need for a measurement method (equimolar reaction) in which the reactivity to each is equal regardless of the abundance ratio of F-PSA and ACT-PSA in serum. ing.

  In IRMA and EIA methods, it has been reported that F-PSA and ACT-PSA react in equimolar amounts by combining monoclonal antibodies of F-PSA and ACT-PSA (Non-patent Document 1). It is said that it is difficult to equalize the reactivity of F-PSA and ACT-PSA by immunological aggregation measurement method using latex or the like. In EIA and the like, one of the monoclonal antibodies can freely move around in the reaction solution as a labeled antibody (high degree of freedom), while the reactivity with the complex is high. Since both antibodies are immobilized, it is presumed that the degree of freedom is lower than that of labeled antibody alone, and as a result, the reactivity with the complex is limited. Thus, several methods have been proposed for equalizing the reactivity of F-PSA and ACT-PSA in immunological aggregation measurement methods.

  There has been proposed a method in which an antibody against F-PSA that does not react with ACT-PSA is added to a test solution, and then a carrier sensitized with an antibody against PSA is added. This technology allows F-PSA and ACT-PSA to have a similar molecular weight by binding a protein (antibody) having a relatively large molecular weight to a region capable of binding to ACT of F-PSA. In this method, the reactivity of F-PSA to an antibody and the reactivity of ACT-PSA are brought close to each other, and a carrier sensitized with an antibody to PSA and both are measured by equimolar reaction (Patent Document 1).

  Two types of monoclonal antibodies that react with F-PSA or ACT-PSA but do not compete with each other and another monoclonal antibody that does not react with F-PSA but reacts with ACT-PSA are separately sensitized to an insoluble carrier, and T-PSA And an immunoagglutination measurement method for measuring ACT-PSA (Patent Document 2). This technology uses latex that has been sensitized with three types of monoclonal antibodies in combination (mixed) with two specific types, so that measurement is not affected by the abundance ratio of F-PSA and ACT-PSA. We have completed a reagent that can be used and a reagent specific only to the complex. However, in this method, monoclonal antibodies that have no change in binding ability in both free and complex forms are selected. As a result, ACT-PSA has only about 80% reactivity compared to 100% of F-PSA. Not obtained.

Tamay TA "Urology" 1995, 45, p729-744 JP-A-2001-311733 Japanese Patent Laid-Open No. 2001-108681

  The present invention provides a novel immunological agglutination measurement method and a reagent for measurement capable of accurately measuring T-PSA in a biological sample, particularly serum, irrespective of the abundance ratio of F-PSA and ACT-PSA. Objective.

  As a result of intensive studies to solve the above problems, the present inventor has different reactivity to F-PSA and ACT-PSA in an immunological aggregation measurement method using insoluble carrier particles such as latex. A measuring method using a monoclonal antibody and a measuring reagent used therefor have been found, and the present invention has been completed.

That is, the present invention has the following configuration.
(1) A method for detecting or measuring an antigen by reacting an antibody sensitized to an insoluble carrier particle with an antigen and observing the aggregation of the insoluble carrier particle caused by the antigen-antibody reaction, wherein Using insoluble carrier particles sensitized with a monoclonal antibody that reacts with both the complex type bound to the molecule capable of binding to the free type and has a stronger binding force to the complex type than the free type And immunological aggregation assay.
(2) The dissociation constant (KD) of the monoclonal antibody is smaller in the complex type bound to the molecule capable of binding to the free form than in the free form of the antigen to be measured (1) Measurement method.
(3) The ratio of the dissociation constant (KD) of the monoclonal antibody to the free form of the antigen to be measured is 0.1 to 0.25. (1)-(2) measuring method characterized by these.
(4) The immunological aggregation measurement method according to any one of (1) to (3), wherein the antigen to be measured is a prostate specific antigen.
(5) A reagent for detecting or measuring an antigen by reacting an antibody sensitized to an insoluble carrier particle with an antigen and observing the aggregation of the insoluble carrier particle caused by the antigen-antibody reaction, comprising a free form of the antigen to be measured Immunological aggregation comprising insoluble carrier particles sensitized with a monoclonal antibody that reacts with both the complex type bound to the molecule capable of binding to the free form and has a stronger binding force to the complex type than the free form Reagent for measurement.
(6) The dissociation constant (KD) of the monoclonal antibody is smaller in the complex type bound to the molecule capable of binding to the free form than in the free form of the antigen to be measured (5) Reagent for measurement.
(7) The ratio of the dissociation constant (KD) of the monoclonal antibody to the free form of the antigen to be measured is 0.1 to 0.25. (5)-(6) measuring reagent characterized by these.
(8) The reagent for measuring immunological aggregation according to (5) to (7), wherein the antigen to be measured is a prostate specific antigen.
(9) The reagent for immunological aggregation measurement according to (5) to (8), wherein the insoluble carrier particles are latex.

  By the method of the present invention, T-PSA in a biological sample, particularly serum, can be accurately measured regardless of the abundance ratio of F-PSA and ACT-PSA. Hereinafter, the present invention will be described in more detail.

  Normally, two types of monoclonal antibodies that bind to PSA as a measurement object are prepared, and these are monoclonal antibodies that have different epitopes and bind to both F-PSA and ACT-PSA. At this time, the first monoclonal antibody used has a particularly strong binding force to ACT-PSA compared to F-PSA, that is, the dissociation constant for ACT-PSA is lower than that for F-PSA. -A monoclonal antibody having a PSA / F-PSA (dissociation constant ratio) of 0.25 or less is selected. In addition, the binding force to each of the separately used monoclonal antibodies may be equal to both, or conversely strong against F-PSA, and is not particularly limited.

  The monoclonal antibody used in the present invention can be obtained by a known method. That is, immunized animals such as mice and rats are immunized in the same manner as polyclonal antibody immunization, and when antibody titer increases are confirmed, antibody-producing cells are collected. The collected antibody-producing cells are made into hybridomas by cell fusion with myeloma, or transformed by EBV transform or the like and immortalized. An antibody producing strain can be established by screening this with an antibody producing ability and further cloning a strain producing an antibody having a necessary activity. When an antibody-producing strain is inoculated into the abdominal cavity of an immunized animal such as a mouse and cultured, a monoclonal antibody can be obtained as ascites. In addition, a monoclonal antibody can be produced by culturing a large amount of an antibody-producing strain in vitro.

  As the insoluble carrier particles for sensitizing the monoclonal antibody used in the present invention, latex particles suitable for being measured by a so-called general-purpose type or a dedicated type automatic analyzer that can measure biochemical items are useful. . Further, examples of the material of the latex particles include polystyrene, styrene-butadiene polymer and the like, and those made of polystyrene which are widely used in immunological aggregation measurement methods are preferable, but are not particularly limited as long as they are suitable for antibody sensitization. The particle diameter of the latex is preferably 50 to 700 nm that is usually used. Furthermore, regarding the uniformity of the particle diameter, latex having a uniform particle diameter or a mixture of latexes having different particle diameters can be used, but is not particularly limited.

  Sensitization of antibodies to latex particles can be performed using known techniques. Usually, since the surface of polystyrene is hydrophobic, physical adsorption to the latex surface is possible. If latex particles having an amino group or a carboxyl group are used on the surface, the antibody can be bound to the surface of the latex particles by chemical binding using glutaraldehyde or a carbodiimide reagent.

  The measuring method used in the present invention is an aggregating measuring method, but the measuring operation can be performed by a known method. For example, optical measurement can be achieved by reacting a biological sample such as serum with an antibody sensitized to carrier particles, and measuring transmitted light or scattered light by an endpoint method or a rate method.

  An example of embodiment of the measuring method in this invention is shown. Of the monoclonal antibodies that bind to PSA as the measurement object, the first monoclonal antibody binds to both F-PSA and ACT-PSA and has a stronger binding force to ACT-PSA than F-PSA. Sensitize to latex particles as The first monoclonal antibody sensitized to the latex particles is brought into contact with a biological sample such as serum. The sensitized monoclonal antibody is then contacted with another latex particle that binds to both F-PSA and ACT-PSA at a different site than the first monoclonal antibody. If the measurement object is present in the biological sample, latex aggregation caused by the antigen-antibody reaction is optically measured.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1. Anti-PSA monoclonal antibody binding force to antigen (1) Immobilization of anti-mouse IgG Fc rabbit antibody on sensor chip (CM5) The carboxyl group of carboxymethyldextran on the sensor chip of BiacoreX (Biacore) for affinity measurement It was activated with NHS (N-hydroxysuccinimide) for 7 minutes, and an anti-mouse IgG Fc rabbit antibody was immobilized using EDC [N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride] as a condensing agent.
(2) Binding of sample antibody To the anti-mouse IgG Fc rabbit antibody-immobilized sensor chip prepared in (1), 5 μL of 50 μg / mL of anti-PSA antibody G3 (or anti-PSA antibody P2) obtained as usual was applied, Coupled to sensor chip.
(3) Binding of PSA antigen 50 μL of 10 nM PSA antigen F-PSA (or ACT-PSA) was applied to the sensor chip to which the antibody was bound in (2), and a sensorgram (response curve) for 4 minutes. Was monitored.
(4) Repeat operation 10 μL of 10 mM NaOH aqueous solution is applied to the sensor chip used in (3), and the anti-PSA monoclonal antibody and the PSA antigen bound in the operations of (2) and (3) are peeled off from the sensor chip. After washing, it was regenerated. Subsequently, the same operations (2) to (4) were performed on the 20, 40, 80, and 160 nM PSA antigens.
(5) Calculation of affinity The dissociation constant, that is, affinity (KD) was obtained from the sensorgram obtained in (3) and (4) above using the analysis software attached to the apparatus.

(6) Results Table 1 shows the results. A smaller KD value indicates a higher binding force to the antigen. From the ratio of the KD values, it can be seen that the monoclonal antibody G3 has about 5 times higher binding force to ACT-PSA than F-PSA. On the other hand, it can be seen that the monoclonal antibody P2 has about two times higher binding force to F-PSA than ACT-PSA.

Example 2 1. Measurement of immunological aggregation using anti-PSA monoclonal antibody and evaluation of antibody reactivity Preparation of Reagent (1) Preparation of Anti-PSA (Prostate Specific Antigen) Mouse Monoclonal Antibody (No. G3) (hereinafter “Anti-PSA Antibody G3”)-Supported Latex Suspension Average Particle Size 0.162 μm, Solid Content 10% w / A polystyrene latex solution of v (hereinafter, “% w / v” is abbreviated as “%”) was diluted with purified water to prepare a 0.2% latex solution. The latex-sensitive antibody solution was prepared by diluting the anti-PSA antibody G3 with an antibody dilution buffer so that the protein concentration was 100 μg / mL. While stirring 1 mL of 0.2% latex solution at room temperature, 0.8 mL of antibody solution was quickly added and incubated at 37 ° C. for 1 hour. Thereafter, 0.2 mL of a blocking buffer was added, further incubated at 37 ° C. for 1 hour, and then centrifuged at 4 ° C. and 60,000 rpm for 20 minutes. The supernatant containing unadsorbed antibody was removed, and 2 mL of a washing buffer solution was added to the resulting precipitate, followed by stirring and washing. Thereafter, centrifugation and washing operations were repeated twice in the same manner to wash the precipitate. Finally, 2 mL of blocking buffer was added to the precipitate, and after stirring well, a dispersion treatment was performed with ultrasonic waves to prepare a 0.1% anti-PSA antibody G3-supported latex suspension.
(2) Preparation of anti-PSA (prostate specific antigen) mouse monoclonal antibody (No. P2) (hereinafter “anti-PSA antibody P2”)-supported latex suspension (1) 0.1% anti-PSA antibody in the same manner A P2 supported latex suspension was prepared.
(3) Preparation of first reagent for PSA measurement A solution obtained by diluting a 0.1% anti-PSA antibody G3-supported latex suspension 4 times (0.025%) with a diluent for specimen dilution is R (G3)- It was set to 1. In addition, as comparative example 1, liquid R (BL) -1 using only the diluted solution, and as comparative example 2, 0.1% anti-PSA antibody P2-supported latex suspension was diluted 4 times (0.025%). Liquid R (P2) -1 was prepared.
(4) Preparation of Second Reagent for PSA Measurement A solution obtained by diluting a 0.1% anti-PSA antibody P2-supported latex suspension 4 times (0.025%) with a diluent for specimen dilution is R (P2)- 2. As Comparative Example 1, 0.1% anti-PSA antibody G3-supported latex suspension and 0.1% anti-PSA antibody P2-supported latex suspension were each diluted 2-fold with a diluent and mixed in the same amount. Liquid R (GP) -2, and as Comparative Example 2, liquid R (G3)-in which 0.1% anti-PSA antibody G3-supported latex suspension was diluted 4-fold (0.025%) with the diluent. 2 was also prepared.

2. Evaluation of Reactivity of Measuring Reagent As described above, it is important to react equimolarly with F-PSA and ACT-PSA in the measurement of PSA. Here, purified F-PSA and ACT-PSA obtained from Stanford University were each diluted to 10 ng / mL and mixed at different ratios (F-PSA: ACT-PSA = 10: 0, 8: 2, 6: 4, 4: 6, 2: 8 and 0:10), the reactivity of F-PSA and ACT-PSA was examined.
(1) Measurement An automatic analyzer TBA-120FR (manufactured by Toshiba Medical) was used for measurement. In this apparatus, after the sample (12 μL) and the first reagent (90 μL) are dispensed, the measurement is performed by dispensing the second reagent (90 μL) about 296 seconds later. From the absorbance obtained by subtracting the average value of absorbance at 317 seconds and 335 seconds after the first reagent dispensing (same measurement wavelength) from the average value of absorbance at 569 seconds and 587 seconds after dispensing one reagent (measurement wavelength: 572 nm) Calculated.

(2) Results Table 2 shows the results. The example is a system using G3 supported latex suspension R (G3) -1 as the first reagent and P2 supported latex suspension R (P2) -2 as the second reagent. Comparative Example 1 is a system in which diluent R (BL) -1 is used as the first reagent, and mixed liquid R (GP) -2 of G3 and P2-supported latex suspension is used as the second reagent. 2 is a reagent composition opposite to that used in the examples, and a system using a P2-supported latex suspension R (P2) -1 as the first reagent and a G3-supported latex suspension R (G3) -2 as the second reagent. It is. In Comparative Examples 1 and 2, when F-PSA is 100% concentration, it is measured as 10 to 11 ng / mL, but the measured value decreases as the ratio of ACT-PSA increases, and when ACT-PSA is 100% concentration Is 7-8 ng / mL. That is, only about 65 to 75% is measured as a relative value with respect to 100% F-PSA concentration. This suggests that G3-supported latex is used as the second reagent, but there is not enough time to react sufficiently with ACT-PSA. On the other hand, in the system using a latex reagent carrying a monoclonal antibody (G3) having high binding force with ACT-PSA as the first reagent as in the example, the mixing ratio of F-PSA and ACT-PSA is not limited. An almost constant value (the above relative value of 90% or more) was exhibited. Therefore, by using a latex reagent sensitized with an antibody having a strong binding force to ACT-PSA as the first reagent, a measurement system showing almost the same reactivity to F-PSA and ACT-PSA can be completed. .

Claims (9)

  A method of detecting or measuring an antigen by reacting an antibody sensitized to an insoluble carrier particle with an antigen and observing the aggregation of the insoluble carrier particle caused by the antigen-antibody reaction, wherein the free form of the antigen to be measured and its release Immunity characterized by the use of insoluble carrier particles sensitized with a monoclonal antibody that reacts with both the complex type bound to the molecule capable of binding to the mold and has a stronger binding force to the complex type than the free type Agglutination assay.   2. The method according to claim 1, wherein the dissociation constant (KD) of the monoclonal antibody is smaller in the complex type bound to the molecule capable of binding to the free form than in the free form of the antigen to be measured. .   The ratio of the dissociation constant (KD) of the monoclonal antibody to the free form of the antigen to be measured is 0.1 to 0.25. The measuring method according to claim 1 or 2.   The immunological aggregation measurement method according to claim 1, wherein the antigen to be measured is a prostate-specific antigen.   A reagent for detecting or measuring an antigen by reacting an antibody sensitized to an insoluble carrier particle with an antigen and observing the aggregation of the insoluble carrier particle caused by the antigen-antibody reaction. A reagent for measuring immunological agglutination comprising insoluble carrier particles that react with both a complex that binds to a mold and a complex that binds to the mold and that is sensitized with a monoclonal antibody that has a stronger binding force than the free form .   6. The measurement type according to claim 5, wherein the dissociation constant (KD) of the monoclonal antibody is smaller in the complex type bound to the molecule capable of binding to the free type than in the free type of the antigen to be measured. reagent.   The ratio of the dissociation constant (KD) of the monoclonal antibody to the free form of the antigen to be measured is 0.1 to 0.25. The measuring reagent according to claim 5.   The reagent for measuring immunological agglutination according to claims 5 to 7, wherein the antigen to be measured is a prostate specific antigen. The reagent for measuring immunological agglutination according to claim 5, wherein the insoluble carrier particles are latex.