JPS60188848A - Quantitative determination of carcinoembryonic antigen by enzyme antibody crosslinking method - Google Patents

Abstract

PURPOSE:To increase the specificness and sensitivity of quantitative determination by measuring the amt. of oxygen in the enzyme anti-enzyme antibody complex obtd. by conjugating successively an antibody which recognizes a carcinoembryonic antigen, the carcinoembryonic antigen to be measured, three kinds of specific antibodies and enzyme with an insolubilized solid. CONSTITUTION:An antibody (the 1st antibody) which is any among an antiserum for a carcinoembryonic antigen (CEA), single clonic antibody and a mixture which is composed of the plural single clonic antibodies having different parts for recognizing the antigen and recognizes CEA, the CEA to be measured such as blood plasma, an antibody (the 2nd antibody) which is manufactured of the animal species different from the 1st antibody and recognizes CEA, an antiimmune globulin antibody which is the same animal species as the 2nd antibody and is the antiserum to recognize immune globulin, an antienzyme antibody which is either of the antiserum or single clonic antibody and is the antibody manufactured of the same animal species as the 2nd antibody and enzyme used generally by an enzyme immune method are successively conjugated with an insolubilized solid consisting of a plastic molding. The enzyme anti-enzyme antibody complex is manufacture and the amt. of oxygen thereof is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme immunological method for quantifying carcinoembryonic antigen in a test sample solution.

Quantification of carcinoembryonic antigen (CEA) is meaningful for early detection of various cancer diseases and cancer diagnosis in prognosis management, as blood CEA levels are high in patients with malignant tumors and are closely related to the degree of cancer progression. be.

C: EA was initially considered to be an antigen specific to colorectal cancer, but subsequent research has revealed that it exists not only in colorectal cancer but also in other cancer tissues.
eterogeneity) and tissue specificity.

Conventionally, blood CEA levels have been measured by radioimmunoassay or enzyme immunoassay using anti-CEA blood/# (polyclonal antibody), which is specific enough to identify the diversity and tissue specificity of C,EA molecules. lack of sensitivity and measurement sensitivity.

In view of these circumstances, the present inventors made improvements to increase the specificity and sensitivity of quantification, discovered a highly specific method capable of quantifying CEA even in a low intensity range, and completed the present invention.

The present invention provides: (1) an insolubilized solid or an insolubilized solid treated with coating, (1) an antibody that recognizes CEA (first antibody);
■ CEA to be measured, ■ An antibody that recognizes CEA derived from an animal species different from the first antibody (second antibody), ■ Anti-immunoglobulin antibody that recognizes immunoglobulin of the same animal species as the second antibody, ■ Second antibody A method of placing an enzyme-anti-enzyme-antibody complex obtained by sequentially binding an anti-enzyme antibody prepared using the same animal species as the antibody and an enzyme in a rose vat.

The invention will be explained in detail below. (2) The insolubilized solid is a molded article made of plastic such as polyvinyl chloride, polystyrene, or polyethylene, such as a plate, a container such as a microplate or a test tube, or beads. Among them, polyvinyl chloride microplates are anti-C
It is preferable because it has a high binding capacity for the EA antibody and the washing operation is simple.

Combined waves of anti-CEA antibody and, ultimately, CEA in the test sample solution
Washing the insolubilized solid, or concanavalin A (ConA), in order to increase the coupled waves and increase the sensitivity of sizing
Phytohemagglutinin (phytoagglutinins such as Pf(A),
The insolubilized solid can be coated with a coating agent such as a polysaccharide such as dextran, an amino acid mono-heteropolymer, or a bacterial component such as protein A.

The coating process can be performed, for example, as follows. That is, when the insolubilized solid is a polyvinyl chloride microplate (96 wells), water or phosphate buffered saline (hereinafter referred to as P) is added to the microplate.
PHA-P (E
Manufactured by IY Laboratories, A L-18
The insolubilized solid can be coated with the coating agent by adding 50 to 200 μ6 of a coating agent such as 00 to 500 μt/mtm and incubating at 4 to 87° C. for 15 minutes to 2 hours. Next, after removing the solution containing the coating agent, it is dried at 20 to 87°C, and further washed 1 to 4 degrees with a buffer such as PBS.

■The antibody that recognizes CEA (first antibody) means an antiserum against CEA (a mixture of polyclonal anti-monoclonal antibodies and multiple monoclonal antibodies with different antigen recognition sites) As such a first antibody, it is possible to use an unpurified antibody or an antibody purified by a known biochemical technique such as ammonium sulfate fractionation, ion exchange chromatography, or chromatography. Furthermore, the entire immunoglobulin molecule may be used as the antibody, or F(ab') or fragments may be used.

As this antibody (first antibody), it is necessary to use an antibody derived from an animal species other than the animal species from which the anti-enzyme antibody (described below) is derived.

Antiserum can be obtained by immunizing animals such as rabbits, goats, and sheep with CEA by a known method. A simple anti-CF, A antibody can be used, for example, by Kohler
et, al.

Somatic Ce1l Genetics 9,
3Q3 (1977) Also, patent application No. 57-160516
Anti-C
One step is to create a fused cell (hybridoma) from EA antibody-producing cells and mouse (rat) myeloma cells, clone the hybridoma, and then culture it in a test tube or ascites using an animal. Therefore, it can be obtained.

To bind an anti-CEA antibody to the above-mentioned insolubilized solid or coated insolubilized solid, the following method can be performed, for example.

That is, add 60 to 200 μe of a solution of 10 to 500 μf/− of anti-CEA antibody dissolved in a buffer such as I'BS to a container such as a microplate made of the insolubilized solid,
The anti-CEA antibody is bound to the insolubilized solid by incubation at 20-87°C for 1-5 hours, followed by anti-CEA antibody.
After removing the EA antibody solution, it is dried at 20-87°C.

In addition, add 1 to 5% beef extract, Men rubumin cnsA) m solution.
Added o~2oope.

Anti-C
The portion of the EA antibody that did not bind to the insolubilized solid was blocked with BSA. After removing excess BSA solution, 20-87
Dry at ℃.

By non-specifically binding the anti-CEA antibody to the insolubilized solid as described above, it is possible to specifically bind a large amount of CEA in the test sample via the anti-CEA antibody, and as a result, it is possible to quantify It was possible to increase the sensitivity of

Note that when CEA was nonspecifically bound directly to the insolubilized solid, the number of bound CIA molecules was small and the sensitivity of quantification was low.

■The CEA to be measured is a sample to be measured. Preferred are plasma, serum or urine. Perform pre-processing such as extraction as necessary.

The CEA to be measured can be combined, for example, in the following manner.

Add 50 to 200 μe of the test sample to the insolubilized solid bound to the above anti-CEA antibody, and add 1 to 15 μe of the test sample at 20 to 8.7°C.
CEA in the test sample is bound by incubating for 1 hour or overnight at 4°C or 1-5 hours at 20-87°C followed by overnight at 4°C. After removing the test sample, the sample is washed with a buffer such as PBS.

■The antibody (secondary antibody) that recognizes GEA is an anti-CEA antibody produced in an animal species different from that of the first antibody.\Antiserum (polyclonal antibody), monoclonal antibody, or multiple monoclonal antibodies. Any antibody consisting of antibodies may be used, but
Preferably, either one or both of the first antibody and the second antibody is a monoclonal antibody. Monoclonal anti-C
By using an EA antibody, cross-reactivity between similar molecules can be reduced, specificity can be increased, and specific CEA molecules can be differentially quantified.

The method for producing the antiserum and monoclonal antibody is the same as for the first antibody. Furthermore, not only the whole immunoglobulin molecule but also IF and (ab')2 fragments can be used.

To bind the second antibody, for example, C
The insolubilized solid bound to EA was injected with a buffer containing 5 to 10% normal animal serum and 0.1 to 0.5% TwinzO.
A 0 to 5,000-fold diluted anti-CEA antibody (second antibody) solution was incubated for 5 hours, and then incubated with a buffer such as PBS. In another embodiment, by adding an anti-CEA antibody (second antibody) at the same time as the test sample, test CEA and anti-CEA antibody (second antibody) are washed.
can be reacted sequentially. In this case, the test sample 5 is added to the insolubilized solid bound to the anti-CEA antibody (first antibody).
0-200 μe and 5-10% normal animal serum, 0.1
50-5 in buffer containing ~0.5% Twin [phase] 20,
000 times diluted anti-CEA antibody (second antibody) solution 60
Add ˜200 μe and incubate at 20-87° C. for 1-6 hours, or at 4° C. overnight, or at 20-87° C. for 1-6 hours followed by 4° C. overnight.

(2) Anti-immunoglobulin antibody is an antiserum that recognizes immunoglobulin of the same animal species as the second antibody. By a known method, an animal different from the animal species used for the second antibody is immunized with immunoglobulin from the same animal species as the second antibody to obtain an antiserum. Not only all immunoglobulin molecules but also F(a
b')2 fragments can also be used.

To bind the present anti-immunoglobulin antibody, for example, the insolubilized solid to which the second antibody has been bound as described above is washed 1 to 4 times with a buffer such as PBS, and then mixed with 5 to 10% normal animal serum and 0.1 Add 50 to 200 μ4 of anti-immunoglobulin antibody diluted 60 to 5,000 times with a buffer containing -0.5% Twin 20 and incubate at 20-87°C for 0.5 to 5 hours.

Furthermore, by adding this anti-immunoglobulin antibody to the test sample and the antibody (second antibody) that recognizes the CEA at the same time, it is possible to cause the test CEA, the second antibody, and the anti-immunoglobulin antibody to react in sequence. can. In this case, anti-C
Test sample 50-200μ6.5-10% normal animal serum (or 1
Anti-immunoglobulin antibody 50 diluted 60 to 5,000 times in the above g street solution and 50 to 200 μe of solution in a buffer containing ~5% BSA, ) and 0.1 to 0.5% TwinO20. ~200μe is added simultaneously.

0.6-5 hours at 20-37℃, overnight at 4C, or 20
Incubate at ~87°C for 0.5-5 hours followed by overnight at 4°C.

As the anti-immunoglobulin antibody, a mixture of an anti-immunoglobulin antibody chemically bound to and labeled with capsule 0 described below and an unlabeled anti-immunoglobulin antibody can be used. In this case, 50 to 200 μg of an enzyme-labeled anti-immunoglobulin antibody diluted 10 to 10 times with a buffer containing 5 to 10% normal animal serum and 0.1 to 0.5% Twin■20, and the above buffer. At the same time, add 20 to 200μ4 of an unlabeled anti-immunoglobulin antibody diluted 50 to 5,00.0 times,
Incubate at 20-87°C for 0.5-5 hours.

This anti-immunoglobulin antibody is interposed between the anti-CEA (second antibody) and the anti-enzyme antibody described below to crosslink them. This crosslinking is performed using an antigen-antibody reaction. This binding method (enzyme-antibody crosslinking method) has extremely mild reaction conditions compared to conventional methods of binding enzymes to antibodies using chemical reactions, and can avoid a decrease in enzyme activity in the chemical binding method.

Furthermore, it is thought that the number of anti-immunoglobulin antibodies that bind to one molecule of anti-CEA antibody is not limited to one molecule, but that many molecules bind, and moreover, the number of anti-diamond antibodies that bind to one molecule of anti-CEA antibody is also one molecule. A large number of molecules are bound together. That is, the number of enzyme molecules that bind to the CEAI molecules in the test sample was amplified, and the sensitivity of quantification was extremely increased.

■Anti-enzyme antibody is an antibody that recognizes the enzyme used as described below. Antiserum (polyclonal antibody), single clone 1
Any antibody may be used, but it must be produced in the same animal species as the second antibody. As this antibody,
Not only all immunoglobulin molecules but also F (a b')
Two fragments can also be used.

Antiserum is obtained by immunizing an animal of the same species as the second antibody with the enzyme to be used (described later) by a known method.

Monoclonal antibodies can be prepared using known methods (e.g. Kljhl
er et al,, Somatic Ce1l Ge
The hybridoma is obtained by fusing anti-enzyme antibody-producing cells with myeloma cells and culturing the resulting hybridoma in a test tube or by incubating it in animals.

The binding of the anti-enzyme antibody can be carried out, for example, by incubating the insolubilized solid to which the anti-immunoglobulin antibody has been bound in a buffer solution such as PBS for 1 to 4 hours.
After washing twice, 5-10% normal animal serum and 0.1-0.5%
lOo ~ 5,00 in a buffer containing twin [phase] 2o
Add 50 to 200 μa of 0-fold diluted anti-enzyme antibody,
This is done by incubating at 20-87°C for 10 minutes to 2 hours.

■Enzyme means an enzyme commonly used in enzyme immunotherapy. Examples include peroxidase, alkaline phosphatase, and β-lactosidase. To bind the enzyme, for example, the insolubilized solid to which the anti-enzyme antibody has been bound is washed 1 to 4 times with a buffer solution, and then the insolubilized solid with the anti-enzyme antibody bound thereto is washed with a buffer solution for 0.1 to 4 times.
Enzyme solution diluted to a concentration of 10μt/gl 50"200
This is done by incubating μ6tJO at 20-87° C. for 10 minutes to 2 hours. By adding this enzyme at the same time as the anti-enzyme antibody, the anti-enzyme antibody and the enzyme can be bound sequentially. In this case, the insolubilized solid to which the anti-immunoglobulin antibody has been bound is mixed with 50 to 200 μe of the anti-enzyme antibody diluted 1 to 4 times 5.000 times with a buffer such as PBS and 0.0 μe of the anti-enzyme antibody with the above buffer. 1~10μ
Add 50 to 200 μe of enzyme solution diluted to a concentration of t/d, and incubate at 20 to 87° C. for 10 minutes to 2 hours.

Measurements on bound enzymes are performed by reacting with the corresponding substrate, usually
Spectroscopic methods measure the absorbance at the wavelength corresponding to the substrate. The measured binding enzyme activity reflects the CEAfi present in the test sample solution. That is, a known quantity of C
The relationship between the amount of enzyme that binds to EA is determined separately using a calibration curve.
The amount of CEA to be tested is determined using this calibration curve.

When azinobis(8-ethylbenzothiazolin-6-sul)onic acid) (ABTS) is used as a substrate, the absorbance at a wavelength of 405 nm is measured.

As described in detail above, first, anti-CE is added to the insolubilized solid.
A. To efficiently bind CEA in a test sample solution via the antibody, and secondly, to detect CEA in a test sample sensitively and stationarily, anti-CEA antibodies and anti-enzyme antibodies are required. The basis of the present invention is to determine the amount of enzyme that binds chestnut, coriander, and anti-enzyme antibodies using an anti-immunoglobulin antibody (measured antibody cross-linking method). Further, when a mixture of an enzyme-labeled substance and an unlabeled substance is used as the anti-immunoglobulin antibody, the quantitative sensitivity can be further increased.

According to the present invention, the quantitative sensitivity can be increased, and it is possible to determine CEA in a low concentration region present in a test sample liquid.

This indicates that by binding the anti-CEA antibody to the insolubilized solid, more CEA in the test sample solution could be bound to the insolubilized solid, and that a method widely used in conventional enzyme immunoassays In other words, instead of the method of directly binding an enzyme to an antibody through a chemical reaction, a method of binding an enzyme to an antibody using an antigen-antibody reaction (enzyme-antibody crosslinking method) is used to bind one molecule of CEA. This is thought to have been achieved by increasing the number of arsenic molecules and preventing a decrease in enzyme activity due to chemical bonding. The sensitivity for determination of CEA by the conventional method was 1 nf/ml or more, but in the present invention, it was 0.067 nf/g/ or more.

Furthermore, in the present invention, by using a monoclonal antibody as the anti-CEA antibody for at least either the first antibody or the second antibody, cross-reactivity between CEA-like molecules is reduced,
We were able to increase specificity.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Rabbit anti-CEA antiserum (I
gG purified product, manufactured by QAKO) 1 ooμe of an aqueous solution of 1 otit/ml was added per well, and the mixture was incubated at 87° C. for 2 hours. Remove the above aqueous solution and then
After drying for 10 minutes, 8% bovine serum albumin (BS
Processing prss2o0ttll containing (abbreviated as A), 87C
The adsorbed part of the anti-CEA antiserum was blocked with BSA by reacting for 1 hour. After removing the above buffer solution, drying at 87°C for 10 minutes, and washing 8 times with PBS, 100 fi of the test sample solution containing CEA to be quantified was prepared.
e Mouse monoclonal anti-CEA antibody diluted 260 times in PBS containing Oyohi 5SB S A, 0.1% Twin■20 (manufactured by Hybritech; mouse ascites purified product;
1: IW/*/ ) 20 μ6 It was added and incubated overnight at 4°C. After removing the reaction solution and washing three times with PBS, 100 μe of rabbit anti-mouse immunoglobulin antibody diluted 100 times with PBS containing 10% normal rabbit serum and 0.1% Twin 20 was added and incubated at 87°C.
Incubated for minutes. After washing 8 times with J''BS, 100μ4 of a mixture consisting of mouse anti-peroxidase antiserum diluted 1.000 times with the above buffer and peroxidase diluted to 1 μt/d with the above buffer was added and the mixture was heated to 87°C. After removing the mixed reaction solution and washing 8 times with PBS, the mixture was prepared at a concentration of 12.5 μ/d with a citric acid-phosphate buffer containing sodium perborate. Azinobis(8-ethylbenzothiazoline-6-sulfoheptaic acid) (ABTS omitted under J2L)
The mixture was allowed to react for a minute to develop color. The absorbance at a wavelength of 405 nm was measured and compared with the detection line to determine the cwhH in the test sample solution. For the calibration curve, 5-wing BSA was used instead of the above test sample solution.
, standard CE dissolved in PBS containing 0.1% Twin20
A was measured and created as described above. Table 1 shows the values of the calibration curve.

Table 1 ☆ (Pack grand value A405 = 0.207 corrected) As a result, by the quantitative method based on the present invention, O, '2
CEA of n f/wtl or more could be measured with good reproducibility, and the measurement sensitivity was significantly improved.

Example 2 Rabbit anti-CEA antiserum (I
gG purified product, DAKO) 100pP/resistant aqueous solution
Add 100 μe per well and incubate at 87° C. for 2 hours. After removing the above aqueous solution and drying at 87°C for 10 minutes, 8% bovine serum albumin (hereinafter abbreviated as BSA) was added.
By adding 200 μ6 of rPBs and reacting at 37°C for 1 hour, the part of the anti-CEA antiserum that was not adsorbed was absorbed into B.
Blocked with SA. Remove the above buffer and store at 87°C for 1
After drying for 0 minutes and then washing 8 times with PBS, the test sample solution containing CEA to be quantified was 100 pH Oyohi 5% B S
A, 0.1%7 (25 on PBS including N20)
0-fold diluted mouse monoclonal anti-CEA antibody (manufactured by Hybritech; mouse ascites purified product; 'gG
x : IW/g/ ) 20 P(l wo, 4C
I incubated it overnight. Remove the reaction solution, PBS
After washing three times at 10% commercial rabbit serum and 0.1% Twin 20, add 100 μe of rabbit anti-mouse immunoglobulin antibody diluted 100 times with BS at 37°C.
Incubated for 30 minutes. After washing three times with PBS,
Mouse anti-peroxidase antiserum diluted i, ooo times with the above buffer and 1 liter / 1 liter with the above buffer.
d, a mixture consisting of peroxidase diluted in 1
00μ4 was added and incubated at 87°C for 80 minutes.

The mixed reaction solution was removed, washed 8 times with 1"Bs, and washed 12 times with a citric acid-phosphate buffer containing sodium perborate.
．． Azinobis (
Add 100μ4 of 3-ethylhendithiazoline-6-sulfonic acid (ABTS) substrate solution and incubate at 87℃ for 8 hours.
The mixture was reacted for 0 minutes to develop color. The absorbance at a wavelength of 4Q5 nm was measured and compared with a calibration curve to determine the amount of CEA in the test sample solution. For the calibration curve, 5% B5 was used instead of the above test sample solution.
A1 Q was prepared using standard CEA dissolved in PBS containing 1% Twin 20 and measured as described above. Table 2
shows the values of the calibration curve.

Table 2 CEA (nr/ag/) 0.0670.2 0.6
1.8 Measurement average value (A405) 0.828 0.59B
0.829 1.025*(background value A40
5 = 0.218 corrected) As a result, by the quantitative method based on the present invention, CEA of 67 pf/1 g1 or more could be measured with good reproducibility, and the measurement sensitivity was significantly improved.

Claims (1)

[Scope of Claims] (1) An insolubilized solid or a coated insolubilized solid contains an antibody (first antibody) that recognizes carcinoembryonic antigen, a carcinoembryonic antigen to be measured, and an animal different from the first antibody. An antibody (second antibody) that recognizes species-derived carcinoembryonic antigen, an anti-immunoglobulin antibody that recognizes immunoglobulin of the same animal species as the second antibody, and an antibody produced using the same animal species as the second antibody A method for quantifying carcinoembryonic antigen by measuring the amount of enzyme in one enzyme-antienzyme complex obtained by directly binding an anti-enzyme antibody and an enzyme. (2) The carcinoembryonic antigen according to claim 1, wherein either or both of the antibodies (the first antibody and the second antibody) that recognize the carcinoembryonic antigen are monoclonal antibodies. How to quantify. (8)' The anti-immunoglobulin antibody that recognizes the immunoglobulin is a mixture of an anti-immunoglobulin labeled by chemically binding an enzyme and an unlabeled anti-immunoglobulin antibody; 3. A method for quantifying carcinoembryonic antigen according to item 2.