JPH02264864A - Immunoassay reagent of gsa-2-binding protein and immunoassay kit using the same - Google Patents

Abstract

PURPOSE:To measure GBP accurately and rapidly by immobilizing either of an antibody to glycoprotein (GBP) having bindability to GS-2 reacting with nonreducible terminal N-acetylglucosamine and GSA-2 on a solid phase carrier and combining the other with a reagent labeled with labeling substance. CONSTITUTION:GSA-2 (Griffonia simplifolia aggulutinin-2) specifically reacting with nonreducible terminal N-acetylglucosamine and GBP having bindability to GSA-2 are used as principal constitutional elements and one of them is immobilized on a solid phase carrier and the other is combined with a reagent labelled with detectable labeling substance. Herein, as a component immolibilizing one of said constitutional elements on the solid phase carrier of a measuring reagent, anti-GBP is desirably used. As the solid phase carrier, a high-molecular compound such as polystyrene or polyethylene is used. Further, as the labeling substance, enzyme or fluorescent substance can be used.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to GSA-2 (Griffoniasi
+nplifolia agglutinin-2)
The present invention relates to a binding protein immunoassay reagent and an immunoassay kit using the same.

[Conventional technology]

Studies using monoclonal antibodies have revealed that as cells become malignant, changes occur in the sugar chain structures of glycoproteins and glycolipids present on the cell surface. By detecting tumor-associated sugar chain antigens with altered sugar chains, histological and serological diagnosis of cancer becomes possible.
) l 9-9 and sialyl 5SEA-1 (some tumor-associated carbohydrate antigens are used for diagnosis. These tumor-associated carbohydrate antigens consist of galactose (Gal) and N-acetylglucosamine (GlcNAc). It has a basic structure, which is fucose (Fuc) and sialic acid (NeuAc).
Many of them are modified by CA19-9 and sialyl 5SEA-i also have such a structure. These tumor-associated carbohydrate chain antigens are immunized in mammals such as mice, rats, rabbits, goats, or sheep to create specific antibodies against these antigens, which are then used to detect these antigens histologically or serologically. Cancer is diagnosed by detecting cancer.

[Problem to be solved by the invention]

However, cancerous changes in sugar chain antigens are extremely diverse depending on the site of cancer, tissue type, differentiation, etc. Therefore, it is difficult to diagnose all cancers using one tumor-related sugar chain antigen. As a method to compensate for this drawback, multiple types of tumor-related antigens have been developed, and attempts are being made to combine them to increase the efficiency of cancer diagnosis. However, the current situation is that it is still difficult to say that either of these is sufficient.

[Means to solve the problem]

In view of this problem, the present inventors aimed to discover new tumor-related carbohydrate antigens and further improve the efficiency of cancer diagnosis.
I considered it carefully. As a result, GSA-2 (Griffonia simplifi
The present invention was achieved by discovering that sugar chain antigens that specifically react with Olia agglutinin-2 are present at high frequency on the surface layer of cancer cells. That is, the first invention of the present invention is directed to a glycoprotein (GBP: GSA-2 Binding
Antibody against Glycoproteinl and GSA-
This is an immunological measurement reagent for GSA-2-binding protein, which is a combination of two main components, one of which is immobilized on a solid phase carrier, and the other of which is labeled with a detectable labeling substance. The second invention of the present invention uses GSA-2 and an antibody against a glycoprotein that specifically reacts with non-reducing end N-acetylglucosamine and has binding properties to GSA-2 as main components, and either one is placed on a solid phase. Immunization of GSA-2 binding proteins by combining a measurement reagent that is immobilized on a carrier and labeled with a detectable labeling substance, and a standard material for creating a calibration curve, a solubilizer, and a detergent. This is a kit for scientific measurement. A third invention of the present invention is that the labeling substance described in the second invention is an enzyme, and a substrate for measuring the enzyme activity and a reaction terminator for the enzyme are added to the immunoassay kit of the second invention. This is a kit for immunological measurement of GSA-2 binding protein. The component to be immobilized on the solid support of the measurement reagent is an anti-GBP antibody or GSA-2, but it is preferable to use an anti-GBP antibody because antibodies have higher reaction specificity than lectins. As the solid support, for example, in addition to polymeric compounds such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, fluororesin, and polysaccharide, paper, glass, metal, and combinations thereof can be used. As for the shape of the solid phase carrier, various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a test tube, etc. can be used. The labeling reagent, which is another main component of the measurement reagent, is a labeling reagent in which an anti-GBP antibody or GSA-2 is bound to a labeling substance. As this labeling substance, enzymes, fluorescent substances, luminescent substances, radioactive substances, etc. can be used. Examples of enzymes include peroxidase, alkaline phosphatase, and β-D-galactosidase. Examples of fluorescent substances include fluorescein isothiocyanate and phycobiliprotein. Examples of luminescent substances include isoluminol, lucigenin, and acridinium salts. For example, 1″II for radioactive substances,
+s+1.3*p, 3H, etc. These are not limited to the exemplified ones, and others can also be used as long as they can be used in immunoassays. When using an enzyme as a labeling substance, a substrate and, if necessary, a coloring agent are used to measure its activity. When peroxidase is used as the enzyme, hydrogen peroxide is used as the substrate, and 2.2% is used as the coloring agent.
°-Azinodi-[3-ethylbenzthiazolinesulfonic acid ammonium salt] (ABTS), 5-aminosalicylic acid, 0-phenyl diamine, 4-aminoantipyrine, 3.3°, 5,5° tetramethylbenzidine, etc. Use. When alkaline phosphatase is used as the enzyme, for example, 0-nitrophenyl phosphate or the like is used as the substrate. When using 110-galactosidase as the enzyme, for example, fluorescein enzyme (t
J-D-galactopyranoside), 4-methylumbe J-ferriruto D-galactopyranoside, and the like can be used. The above-mentioned anti-GBP antibody can be obtained as an antibody component of anti-GBP antiserum obtained by immunizing an animal with GBP as an antigen by a known method, or a fragment thereof. Animals used for immunization include rabbits, goats,
Sheep, mice, rats, etc. are suitable. GBP can be separated by homogenizing human colon cancer cells, treating them with a solubilizing agent, and then performing gel filtration to collect a fraction that is reactive with GSA-2.

[Action and effect of the invention]

To measure GBP in a specimen using the measurement reagent or compound of the present invention, the reagent bound to a solid phase carrier, a specimen sample diluted with a dissolving agent, etc., and a standard substance of known concentration are separately separated. are brought into contact with and reacted at a pre-temperature for a certain period of time. Next, after separating the reaction solution from the immobilized reagent and thoroughly washing the immobilized reagent, a labeled reagent labeled with an appropriate standard substance is added and reacted for a certain time and temperature. Furthermore, after separating the reaction solution and thoroughly washing the immobilization reagent, the amount of bound labeled reagent is measured, and the GBP concentration in the specimen sample is read from a calibration curve created from the measured values of standard substances with known concentrations. This can be done by By using the measurement reagent or kit of the present invention, it becomes possible to accurately and quickly measure GBP present in serum and the like with high specificity and sensitivity. As a result, it is useful in diagnosing gastrointestinal cancer and the like.

【Example】

Hereinafter, the present invention will be explained in detail with reference to Examples. Note that the percentages in the examples are based on weight. (Separation and purification of GBP) 499.5 g of fresh human colon cancer was homogenized in 500 mj of a chloroform-methanol mixture (volume ratio 2:l), stirred at room temperature for 1 hour, and then filtered through a filter paper. The residue was mixed with a chloroform-methanol mixture (volume ratio: 1
:1) Wash with 100raε and then 200% ethanol
After washing three times with 1.0 mg, the sample was suspended in distilled water and freeze-dried to obtain a sample with a dry weight of 1.25 g. 1.0 g of this sample was added to O, QIIlol phosphate buffered saline with a pH of 7.4, and stirred at 4°C for 24 hours.
After precipitation by centrifugation at 000rpI11° for 60 minutes, add 0.01M phosphate buffered saline 50% to the remaining part.
ml was added and stirred at 4°C for 24 hours, and the supernatant was combined with the supernatant obtained from the above precipitation to give 22,000rp111.3
Precipitation by centrifugation for hours was performed and the resulting residue portion was combined with the previous residue portion. This residual part is l 1lI
ool of dithiothreitol and 25mMol of 3-[
(3-chloramidobrovir)-dimethyl-ammonio]
-50m of 0.01M phosphate buffered saline containing 1-propanesulfonate (CIAPS)! After solubilization at 4°C for 24 hours using
Orpm, precipitate by centrifugation for 2 hours. The residue portion was treated with 1 mMo1 dithiothreitol and 1 mMo1 dithiothreitol.
The mixture is added to 50 g++1 of 0.01 Mol phosphate buffered saline containing 6 mM CHAPS, stirred at 4° C. for 24 hours, and then centrifuged at 30.00 rpm for 2 hours. Both supernatants combined 0.0IMol
Dialysis was performed against phosphate buffered saline. After concentrating it, 0.00 g containing 8 g Mol of CHAPS was obtained. O
Gel filtration was performed using IMol phosphate buffered saline using a Sephacryl S-400 (Pharmacia) column, and lOgj was taken from each half and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodiun).
+ dodecylsulfate-polyacry
lamidegeelectrophoresis:
5DS-PAGE). After the electrophoresis was completed, the proteins were transferred from the slab gel to the nitrocellulose membrane by applying current for 1.5 hours at 25 μm. After blocking this nitrocellulose membrane with 0.01M phosphate buffered saline containing 3% bovine serum albumin, a horseradish peroxidase-labeled GSA-2 solution was added and reacted for 1 hour at room temperature. Next, wash thoroughly and add 0.01Mol containing 0.04% 3.3°-diaminobenzidine and 0.0034% HzO□.
After adding a substrate solution consisting of phosphate buffered saline to develop color, the GSA-2 positive fraction was collected, dialyzed against 0.01M phosphate buffered saline, and lyophilized to obtain 8.6 Obtained GBP of rag. (Preparation of anti-GBP antibody) GB extracted and collected from human colon cancer tissue as described above.
1 mmg of P is 1+s! Suspended in physiological saline and emulsified with Freund's complete adjuvant, a male rabbit (weight 1.9K) was prepared.
g) Immunization was performed by administering the vaccine to the hind paw. After 14 days, 5-g of GBPo was suspended in 0.5 mg of physiological saline and then boosted with antigen emulsified in complete Freund's adjuvant.
Animals were sacrificed on day 1 to obtain antiserum. 50% to the obtained antiserum
The antibody was precipitated by adding a saturated aqueous ammonium sulfate solution, and the precipitate was filtered off and then dissolved in 0.01M phosphate buffered saline. and after dialysis against 0.01M phosphate buffered saline, Protein A-Sepharose CLJB
Column (manufactured by Pharmacia) and apply the antibody to 0.2Mo
The purified anti-GBP antibody was obtained by elution with lglycine-hydrochloric acid buffer (PH3, Q) and neutralization. Example 1 Preparation of GBP measurement reagent and measurement of GBP using the same (1) Preparation of anti-GBP antibody immobilized carrier Into each well of a polystyrene microplate (manufactured by Nunc), rabbit anti-GBP antibody was added at lOxg/ms. Has a concentration of 0. O
It was left in 5Mo1 charcoal j12 buffer (PH9,6) at a temperature of 4°C for one day and night. Set it to 0. 0 containing 1% bovine serum albumin after washing with O5Mo1 carbonate buffer.
Anti-GB
A P antibody-immobilized microplate was obtained. (2) Horseradish peroxidase labeled G
Preparation of SA-2 solution Horseradish peroxidase labeled GSA
-2 (manufactured by EY Laboratories) 0.0IMol
Horseradish peroxidase-labeled GSA-2 was prepared by diluting it 100 times with phosphate buffered saline. (3) Measurement of GBP by Sand-Deutsche method Purified GBP was added to each well of an anti-GBP antibody-immobilized microplate.
(standard substance) in the range of 0 to 100/mj (the concentration that gives an absorption intensity of 1.0 at a wavelength of 492 nm when measured by this measurement method was set to 100/-1).
100 IIj of 0.01M phosphate buffered saline containing % bovine serum albumin was added and cultured at 37° C. for 2 hours. Next, the solution in each well was removed by suction, and the
．． After washing with 01Mol phosphate buffered saline (2
Add 100 ml of the horseradish peroxidase-labeled GSA-2 solution prepared in ) to each well.
Culture was carried out at a temperature of 7°C. The solution in each well was removed by suction l), washed with 11M phosphate buffered saline, then washed with orthophenylenediamine 0.02%, HtO□
Add 100 uffi of 0.1Mo1 phosphate-citrate buffer (PH5.01) containing 0.05% to each well and incubate at 37°C for 30 minutes, then add 100 μl of IN sulfuric acid as a reaction stopper. The absorption intensity of this solution at a wavelength of 492 nm was measured using a microplate league, and by plotting this against the concentration of the standard substance, a calibration curve with good concentration dependence was obtained. The results are shown in Figure 1. Example 2 Measurement of GBP in clinical specimens 10.j of serum from healthy individuals and cancer patients, gastric cancer, pancreatic cancer, and colorectal cancer patients were each collected, and 1% bovine serum was added to the serum. 0.01M phosphate buffered saline containing albumin 90gj
was added to each well of the anti-GBP antibody-immobilized microplate prepared in Example 1 (1), and incubated at 37°C for 2 hours.
Washed with phosphate buffered saline. The horseradish peroxidase-labeled GSA-2 solutions too and j prepared in (2) of Example 1 were added to each well and incubated at a temperature of 37° C. for 2 hours. The solution was removed by suction and washed with 0.01M phosphate buffered saline (after washing, 0.02% orthophenylenediamine, H*0*
Add 10011 of 0.1Mo1 phosphate-citrate buffer (PH5.0) containing 0.05%, incubate at 37°C for 30 minutes, and add 10011 of 5N sulfuric acid as a reaction terminator.
The enzymatic reaction was stopped by adding 11 portions of 0I. The absorption intensity of this solution at a wavelength of 492 nn+ was measured, and the concentration was read using a calibration curve prepared in the same manner as in Example 1. The results were 0.6 U/ml for healthy people (51.60 U/ml for gastric cancer, 13.8 U/ml for pancreatic cancer, 3 U/ml for colorectal cancer).
It was 2.4 U/ml.

[Brief explanation of drawings]

FIG. 1 is a diagram showing a calibration curve for GBP measurement. Patent applicant: Shin-Etsu Chemical Co., Ltd. Figure 1 GBP song/1 regular service GBP drop & (U/m2)

Claims (1)

[Scope of Claims] 1. GSA-2 and an antibody against a glycoprotein that specifically reacts with non-reducing end N-acetylglucosamine and have binding properties to GSA-2 are the main components, and either one is placed on a solid phase. GSA-2, characterized in that it is a combination of reagents immobilized on a carrier and the other labeled with a detectable labeling substance.
Immunological assay reagent for binding proteins. 2. GSA-2 and an antibody against a glycoprotein that specifically reacts with non-reducing terminal N-acetylglucosamine and have binding properties to GSA-2 are the main components, one of which is immobilized on a solid phase carrier, and the other is A kit for immunological measurement of GSA-2-binding protein, which comprises a measurement reagent in combination with a reagent labeled with a labeling substance capable of detecting the same, and a standard material for preparing a calibration curve, a solubilizer, and a detergent. 3. A GSA in which the labeling substance is an enzyme, and a substrate for measuring the enzyme activity and a reaction terminator thereof are added and combined with the immunoassay kit according to claim 2.
-2 binding protein immunoassay kit.