JPH1114626A - Method for measuring ca125 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure CA125 more accurately by immobilizing monohydric and divalent antibodies of immuneglobulin that specifically reacts with CA125 to a solid phase for use. SOLUTION: Adjusted monohydric and divalent antibodies are immobilized to a solid phase for use. For example, red cell, gelatine particles, latex particles, microplate, and test tubes that are normally used in a field concerned may be used as the solid phase. A biological sample that can be measured includes blood constituents such as plasma and serum, thus efficiently reducing interference due to such an interference substance as an antibody (for example, HAMA) for animal immuneglobulin that exists in a measurement sample and maintaining specificity and hence measuring CA125 more accurately. Also, by taking measurement according to a particle clumping method, CA125 can be measured quickly with more simple operation than before.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method of measuring CA125 with high sensitivity by suppressing non-specific reactions.

[0002]

2. Description of the Related Art CA125 is an antigen recognized by monoclonal antibody OC125 and is a glycoprotein having a molecular weight of about 110,000, and is an important test item as a tumor marker for ovarian cancer. It is particularly high in serous cystic adenocarcinoma of ovarian cancer. In addition, CA125 is useful as a marker for gynecological cancer and gastrointestinal cancer as a marker for diagnosis and prognosis because other diseases have high levels in uterine cancer, pancreatic cancer, and gall bladder cancer.

[0003] As a method for measuring CA125, an OC125 antibody is immobilized on a solid phase, reacted with CA125 in a sample, and further reacted with a labeled OC125 antibody, followed by B / F separation and capture by the solid phase. There is a method of measuring the amount of labeled
No. 280061).

However, when a substance that reacts well with the OC125 antibody is present in the measurement sample, for example, when anti-mouse γ-globulin (HAMA) is present in the measurement sample, it is detected that OC
By binding to the 125 antibody, the reaction with CA125, which is originally intended, is inhibited, and accurate measurement cannot be performed. Therefore, countermeasures are being taken using a monoclonal antibody M11 or the like that recognizes an epitope on CA125 different from the OC125 antibody as a labeled antibody. In general, there is also a case where a countermeasure is performed by performing a pretreatment by adding mouse γ-globulin to the measurement sample.

[0005]

However, even if the above-mentioned method is adopted, the interference by anti-mouse γ-globulin (HAMA) is not eliminated, and it is insufficient as a countermeasure method.

[0006] Further, when the measurement is performed by the particle agglutination method, even if two antibodies, OC125 and M11, are sensitized and used on the particles, the particles may aggregate only by the bond of OC125-CA125-M11. Instead, the particles also aggregate due to the bond OC125-CA125-OC125. In such a case, the same problem as described above occurs, and accurate measurement cannot be performed.

Accordingly, an object of the present invention is to provide a method for more accurately measuring CA125.

It is another object of the present invention to provide a method for measuring CA125 using the particle aggregation method.

[0009]

The method for measuring CA125 of the present invention is characterized in that a monovalent antibody and a bivalent antibody of immunoglobulin specifically reacting with CA125 are immobilized on a solid phase and used. I do.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The immunoglobulin specifically reacting with CA125 used in the present invention includes a monoclonal antibody OC.
Including 125 and M11. However, a polyclonal antibody may be used as long as it specifically reacts with CA125, and its origin is not particularly limited. Antibodies can be prepared by a conventional method (for example, Shinsei Chemistry Laboratory Course, Protein I, pp. 389-39).
7, 1992) and immunizing animals with CA125 and collecting antibodies produced in vivo. In the present invention, it is more preferable to use a plurality of antibodies recognizing different epitopes in combination than to use one kind of antibody from the viewpoint of increasing the specificity and sensitivity of the measurement.

The immunoglobulin divalent antibody specifically reacting with CA125 used in the present invention refers to an antibody having two antigen-binding sites in the molecule, for example, a monoclonal antibody
It may be a monoclonal antibody such as OC125 or M11, or an antibody itself such as a polyclonal antibody, or a fragment obtained by subjecting these antibodies to an enzymatic treatment or the like. Such fragments include, for example, F (ab ') ₂ obtained by digesting the antibody with pepsin.

The monovalent antibody of immunoglobulin which specifically reacts with CA125 is an antibody having one antigen-binding site in the molecule, which is obtained by reducing immunoglobulin or subjecting them to enzyme treatment or the like. , Such as Fab and Fab '. A monovalent antibody is obtained by reducing the above-described bivalent antibody to cleave the SS bond. The method for reducing antibodies
Conventionally known methods may be used, for example,
Monovalent antibodies can be recovered by incubating in 1-100 mM 2-mercaptoethanol, 2-mercaptoethylamine, etc. for 1-5 hours.

In the present invention, a monovalent antibody and a bivalent antibody are used by immobilizing them on a solid phase. With a monovalent antibody alone,
Since nonspecific reaction is reduced as well as specificity is reduced, nonspecific reaction due to HAMA or the like can be suppressed while maintaining specificity by using a combination of bivalent antibodies. In the present invention, a divalent antibody can be used after being sensitized to a solid phase to the extent that interference due to nonspecific reaction does not occur. A suitable ratio between the monovalent antibody to be sensitized and the bivalent antibody is 1: 9 to 9: 1, preferably 1: 1 to 1: 5,
An antibody solution mixed in a buffer such as a phosphate buffer solution (PBS) at the above ratio can be used after sensitizing the solid phase. The preferred pH for sensitization is 3-8, more preferably pH 4
~ 6.

In the present invention, the monovalent antibody and the divalent antibody prepared as described above are used by immobilizing them on a solid phase. Any of these can be used, for example, erythrocytes, gelatin particles, latex particles, microplates, test tubes, and the like.

In order to immobilize a monovalent antibody and a bivalent antibody on a solid phase, a method conventionally used for immobilizing a bivalent antibody can be used as it is, by a physical adsorption method. And a covalent bonding method. In particular, the physical adsorption method can be performed by a simple operation. As a general method, for example, the antibody solution obtained above is brought into contact with a solid phase (or mixed with a particle suspension when the solid phase is a particle). ), 4 ℃
The reaction can be carried out overnight or at 25 ° C. for several hours.

The biological sample that can be measured by the method of the present invention contains blood components such as plasma and serum.

In order to measure CA125 according to the present invention,
Any type of immunoassay may be used, including various embodiments of the sandwich method, as well as competition methods. Furthermore, the monovalent antibody and the divalent antibody were sensitized using the particle surface as a solid phase, and the degree of aggregation of the particles was measured to determine CA125.
Can also be measured.

For example, in the sandwich method, a sample containing CA125 is placed in a reaction vessel in which an antibody is immobilized, and incubated at a predetermined temperature for a predetermined time. Next, a labeled antibody solution is added, and further incubated at a predetermined temperature for a predetermined time to cause a reaction. The labeling method for an antibody can be performed by the same method as that for an antibody. In addition,
When an OC125 antibody and a monovalent antibody of OC125 are used as the solid phase antibody, the OC125 antibody or OC125 is used as the labeled antibody.
May be used, but an M11 antibody recognizing an epitope different from OC125 or a monovalent antibody of M11 is preferred. Thereafter, B / F separation is performed to measure the amount of the label captured on the solid phase. Then, the amount of CA125 in the measurement sample is determined from a calibration curve created in advance.

When the measurement is performed using the particle aggregation method, a particle suspension sensitized with a monovalent antibody and a divalent antibody is mixed with a sample containing CA125, and the mixture is mixed at 30 to 50 ° C., preferably 37 to 50 ° C. Incubate at 45 ° C for 5-30 minutes, preferably 10-20 minutes. Thereafter, the state of aggregation is confirmed visually or by a particle counting device. When counting unagglomerated / agglomerated particles using a particle counter (for example, PAMIA series of Toa Medical Electronics Co., Ltd.), it is preferable that the particles used have a uniform particle size. Most preferred. As for the size of the particles, those commonly used can be used, and 0.5 to 1 μm is suitably used. The material is not particularly limited as long as it is used in the art, but polystyrene latex is most preferred.

The antibody sensitizing the particles may be a single kind of antibody. However, if an antibody recognizing another epitope is mixed and sensitized before use, it is advantageous in increasing the specificity and sensitivity. . For example, not only monovalent antibodies of OC125, but also M11
Can be used after sensitization. The ratio of the two antibodies is preferably from 1: 9 to 9: 1, more preferably 1: 1. In order to maintain the specificity, a bivalent antibody corresponding to each monovalent antibody is sensitized and used so as not to cause interference by non-specific reaction with HAMA or the like.
The preferred ratio between the monovalent antibody and the bivalent antibody is as described above. For example, a monovalent antibody and OC125 antibody of OC125 and four antibodies of M11 monovalent antibody and M11 antibody can be sensitized and used on the same particle.

The particle aggregation method is preferable because B / F separation is not required, and the measurement can be performed in a short time with a simple operation.

[0022]

【Example】

Example 1 Preparation of Monovalent Antibody 2.0 mg of OC125 antibody (obtained from Centcore) was added to 10 mM 2-
It is dissolved in 2 ml of a 10 mM PBS (pH 7.0) aqueous solution containing mercaptoethylamine hydrochloride and 0.5 mM EDTA-4Na, incubated at 37 ° C. for 2 hours, and cooled with ice. Thereafter, a 10 mM MPBS (pH 6.0) aqueous solution containing 0.1 M monoiodoacetamide is added so as to be equimolar to the used 2-mercaptoethylamine hydrochloride, and blocked, followed by incubation at room temperature for 2 hours.

Next, Superdex 200pg (Pharmacia)
Perform gel filtration with 10 mM PBS (pH 6.0) to collect monovalent antibodies.

The same operation is performed for the M11 antibody (obtained from Centcor) to recover a monovalent antibody.

Example 2 Preparation of Antibody Sensitized Latex Polystyrene latex having a particle size of 0.8 μm was added to 0.1 M PBS (pH 6.
0) in which a bivalent antibody and / or the monovalent antibody prepared in Example 1 was prepared in latex suspension in various combinations as described below. 300 μg in total per 1 ml of liquid (150 μg in total for OC125 antibody and its monovalent antibody, 150 μg in total for M11 antibody and its monovalent antibody)
The mixture was added and reacted at 4 ° C. for 24 hours. Then centrifuge (12000rp
m, 10 min.), and a 10 mM PBS (pH 6.0) solution containing 5% BSA was added in the same amount as at the beginning to disperse the particles. Another centrifugation treatment was performed and the resultant was dispersed in the same solution to obtain an antibody-sensitized latex. Various latex reagents were prepared with the combinations of antibodies shown in the table below.

[0026]

[Table 1]

[0027]

[Table 2]

Example 3 Measurement of CA125 10 mM PBS (pH 6.0) containing 5% BSA in 10 μl of sample (serum) 80
After adding 10 μl of various antibody-sensitized latex particles (0.5%) prepared in Example 2 and reacting at 45 ° C. for 15 minutes, the aggregation rate of latex particles was measured. This reaction was performed using a fully automatic immunoassay device PAMIA-30 of Toa Medical Electronics Co., Ltd.

The aggregation rate (P / T%) is the total particle count.
It is shown as a count (P) of aggregated particles relative to (T).

With respect to a sample containing CA125 at a known concentration of 0 to 2665 U / ml, the aggregation rate of latex was measured, and the following results were obtained. In addition, CA1 indicating (aggregation rate of latex at 0 U / ml) + 0.2%
The concentrations of 25 were determined from the respective calibration curves, and were used as the sensitivity of the latex reagent.

[0031]

[Table 3]

As is apparent from the results, the sensitivity was remarkably improved by combining (-) with a divalent antibody as compared with () in which only a monovalent antibody was sensitized.

Effect on Non-Specific Reaction Regarding the above items, three specimens exhibiting a non-specific reaction (samples containing a factor causing a non-specific agglutination reaction in a particle agglutination reaction) were measured, and the effect on the non-specific reaction was examined. As a control, RIA method using CA125IIIRMA (using an OC125 monoclonal antibody as a solid phase and radiolabeling an M11 monoclonal antibody as a labeling antibody: Tei Corporation)
As a comparative method, a sensitized latex reagent was prepared by removing a monovalent antibody from the latex reagent as a comparative method, and the same operation was performed to measure CA125.
The results obtained are shown in the table below.

[0034]

[Table 4]

As is clear from the table, in the comparative method, the data becomes high due to the non-specific agglutination caused by the non-specific reaction, but the non-specific reaction is suppressed by combining the monovalent antibody and the bivalent antibody. Is done.

Correlation with the conventional method Using the latex reagent of (1), measurement was performed on 41 samples of patient sera, and the conventional method, CA125IIIRMA (RIA
Method). The regression linear equation and the correlation coefficient are as follows, and a good correlation with the conventional method was recognized. : Y = 1.006X + 2.035, r = 0.965: Y = 1.001X + 3.166, r = 0.974: Y = 1.063X + 2.225, r = 0.965: Y = 1.112X + 4.872, r = 0.960: Y = 1.147X + 5.475, r = 0.961

Example 4: Comparison of latex reagent sensitized only with bivalent antibody and latex reagent of the present invention Latex reagent sensitized with 150 μg of OC125 antibody and 150 μg of M11 antibody (both per 1 ml of latex suspension) Was prepared by the method described above. The correlation between this latex reagent and each of the latex reagents described in Example 3 of the present invention with CA125IIIRMA (RIA method) was determined. 524 samples including those that show non-specific reactions (CA125 concentration up to 500 U / ml)
Was measured.

In particular, in the correlation of a sample (n = 503) up to 140 U / ml, the correlation coefficient r = 0.610 was obtained for the latex reagent containing only a bivalent antibody, whereas the correlation coefficient r was found for the latex reagent of the present invention. The relation number r was 0.884, the nonspecific reaction was suppressed, and the correlation with the RIA method was extremely improved.

[0039]

According to the present invention, interference by an interfering substance such as an antibody (for example, HAMA) against an animal immunoglobulin present in a measurement sample can be efficiently reduced and specificity can be maintained, so that CA125 can be more accurately determined. Can be measured.

Further, by performing the measurement by the particle aggregation method, the measurement of CA125 can be performed in a shorter time by a simpler operation than in the past.

Claims (5)

[Claims] 1. A method for measuring CA125, which comprises immobilizing a monovalent antibody and a bivalent antibody of an immunoglobulin which specifically reacts with CA125 on a solid phase. 2. The ratio of said monovalent antibody to said bivalent antibody is:
The method for measuring CA125 according to claim 1, wherein the ratio is 1: 9 to 9: 1. 3. The CA12 according to claim 1, wherein a plurality of antibodies recognizing different epitopes are used as the antibody.
5 measurement methods. 4. The method for measuring CA125 according to claim 1, wherein the immunoglobulin specifically reacting with CA125 is selected from monoclonal antibodies OC125 and M11. 5. The method for measuring CA125 according to claim 1, wherein the measurement is performed by a particle aggregation method using particles as the solid phase.