JPH0477265B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a sugar antigen, specifically a sugar antigen containing a specific sugar chain as a hapten group, which has specific reactivity with gastrointestinal cancer, etc., and cancer cells, particularly human colon cancer cells and mouse teratocarcinoma cells. , relates to cancer-related saccharide antigens for producing new cancer-related antibodies that are useful in the diagnosis of cancers of the gastrointestinal tract, etc. (particularly colorectal cancer). Recently, at a certain stage of cell differentiation, specific sugar antigens are expressed on the surface of mammalian cells, and monoclonal antibodies that are reactive with such sugar antigens are obtained by cell fusion technology using whole cells as immunogens. Cell.Vol.14, 775−783
(1978), Proc., Natl., Acad., USA, Vol.75,
No.11, 5565-5569 (1978) and Nature, Vol.292,
156-158 (1981)] and antibodies present in the serum of a certain patient [Exp.Cell Res., 131, 185-195 (1981)]
was proposed. In connection with the above reports, the present inventors organically synthesized a specific sugar chain and used it as a hapten group to create a sugar antigen. It is said that it reacts specifically and selectively with cancer cells such as organ cancer, particularly human colon cancer and mouse teratocarcinoma cells, and therefore, by using the antibody, it is possible to recognize and measure cancer cells, and thereby diagnose cancer. I gained new knowledge. The present invention was completed based on this knowledge. That is, the present invention relates to α-fucopyranosyl-(1→3)
The present invention relates to a cancer-associated saccharide antigen consisting of a complex of a carrier protein and an oligosaccharide containing a -, -(1→4)- or -(1→6)-galactopyranosyl group. The production of a fucose antigen, the production of an antibody from the antigen, a cancer diagnostic kit containing the antibody, and a method for measuring cancer-related sugar chains and diagnosing cancer using the same will be sequentially explained below. In the production of the fucose antigen according to the present invention,
α-Fucopyranosyl-(1→3) as a hapten
It is essential to use an oligosaccharide containing a -, -(1→4)- or -(1→6)-galactopyranosyl group. The bond between fucopyranose and galactopyranose, which are essential constituent sugars of the above oligosaccharides, is α1
→3, indicating α1→4 or α1→6 bonds, especially α1→
Three bonds are preferred. Further, the above-mentioned oligosaccharide may further have another sugar chain bonded to its galactopyranosyl group, and a typical example of the sugar constituting the other sugar chain is glycopyranose. The bond between galactopyranose and glucopyranose may be either α or β. Moreover, each of the above-mentioned constituent sugars may be either D-form or L-form. Specific examples of oligosaccharides suitable for the present invention include the following. ●O-α-L-fucopyranosyl-(1→3)-O-
β-D-galactopyranosyl-(1→4)-α-
D-glucopyranose (3'-α-L-fucopyranosyl-α-lactose) O-α-L-fucopyranosyl-(1→4)-O-
β-D-galactopyranosyl-(1→4)-α-
D-glucopyranose (4'-α-L-fucopyranosyl-α-lactose) O-α-L-fucopyranosyl-(1→6)-O-
β-D-galactopyranosyl-(1→4)-α-
D-glucopyranose (6'-α-L-fucopyranosyl-α-lactose) The above oligosaccharide is known or can be easily produced by various known methods [Chem.
Pharm.Bull.29(4)1076-1082 (1981) and 3rd Carbohydrate Symposium Abstracts, pp. 90-91, Title 43
``Synthesis of human milk oligosaccharides'', August 1981]. The above-mentioned oligosaccharide is used as a hapten, and as the carrier protein bound to the hapten, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used. For example, animal albumins such as horse serum albumin, bovine serum albumin (BSA), rabbit serum albumin, human serum albumin, sheep serum albumin, ovalbumin, horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, sheep Animal globulins such as serum globulin and egg globulin, animal thyroglobulins such as horse thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, and sheep thyroglobulin, horse hemoglobulin, bovine hemoglobulin, and rabbit hemoglobulin. , animal hemoglobulins such as human hemoglobulin and sheep hemoglobulin, animal hemocyanins, proteins extracted from roundworms (Ascaris extract, see JP-A No. 16414/1983), edestin, polylysine, poly Examples include glutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine. The reaction between the above-mentioned hapten (oligosaccharide) and carrier protein can be carried out using various known methods such as (A) isothiocyanate coupling method, (B) diazo coupling method,
(C) amide bonding method, (D) reductive amination method, (E) guanidine coupling method, etc. [Advances in Carbohydrate Chemistry and
Biochemistry, Vol.37, p225-281 (1980),
Methods in Enzymology, Vol1, Complex
Carbohydrates, Part C, p155-175 (1978), Protein Nucleic Acid Enzymes Vol.25, No.8, p707-724 (1980)
and Archives of Biochemistry and
Biophysics, Vol.205, No.2, p338−395
(1980)]. The above isothiocyanate coupling method (A
method) involves reacting thiophosgene with a compound produced by a reductive amination reaction (for example, reacting a hapten with a diamine derivative such as β-(p-aminophenyl)ethylamine and a reducing agent such as NaBH ₄ or NaBH ₃ CN). This is then carried out by subjecting the resulting isothiocyanate to a coupling reaction with a carrier protein. The above reductive amination reaction is carried out in a suitable inert solvent such as a buffer such as 0.2M calcium phosphate (PH=8), water, physiological saline, or an alcohol such as methanol or ethanol at 0 to 40°C for 3 hours to 40°C. Progresses well within 3 days. In addition, the reaction between a compound limited to a reductive amination reaction and a thiophosgene can be carried out using an appropriate inert solvent such as water, a 0.1 molar aqueous sodium bicarbonate solution (PH=8), or physiological saline at -10°C to room temperature.
It progresses suitably in 30 minutes to 2 hours. Further, the reaction between the isothiocyanate and the carrier protein is carried out at -10°C in a suitable inert solvent such as water, physiological saline, or 0.1M aqueous sodium bicarbonate solution (PH = 9.5).
Proceeds suitably in ~15-20 hours at room temperature. In the diazo coupling method (Method B), for example, a carrier is added to a diazo compound produced by reacting the compound produced by the reductive amination reaction of the above Method A with a diazotizing agent such as sodium nitrite and hydrochloric acid or sulfuric acid. This is carried out by subjecting proteins to a coupling reaction. The above diazotization reaction preferably proceeds in a suitable inert solvent such as water, physiological saline, or an aqueous mineral acid solution such as an aqueous hydrochloric acid solution at -10 to -20°C for 10 to 60 minutes. Further, the coupling reaction between the diazo compound and the carrier protein proceeds suitably at -10 to 20°C for 2 to 6 hours. The amide bond method (C method) is carried out by, for example, oxidizing the aldehyde group of a hapten with an oxidizing agent such as silver oxide to produce a sugar carboxylic acid, and then subjecting the sugar carboxylic acid and the amino group of a carrier protein to an amide bond reaction. be done. The amide bond reaction can be carried out by a normal peptide amide bond forming reaction, for example, by a dehydration condensation reaction using a dehydrating agent such as 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. This dehydration condensation reaction is carried out using a suitable inert solvent such as 1 molar sodium acetate buffer (PH=5.5).
The process is preferably carried out in a buffer solution such as, for example, at 0° C. to room temperature for 3 to 12 hours. The reductive amination method (D method) is carried out, for example, by reacting a hapten with a carrier protein and a reducing agent such as NaBH ₄ or NaBH ₃ CN. As the conditions for the reductive amination reaction, the conditions for the reductive amination reaction in Method A above can be employed. In the above methods A to D, the amount of each reagent used is at least equimolar to the raw material, and usually preferably in excess. In this way, a desired sugar antigen (fucose antigen) in which an oligosaccharide and a carrier protein are bound can be produced.
The sugar antigen obtained after completion of the reaction can be easily isolated and purified according to conventional methods, such as dialysis, gel filtration, fractional precipitation, etc. Among the sugar antigens obtained as described above, those in which an average of 20 to 25 moles of oligosaccharides are bound to 1 mole of carrier protein are particularly preferred. Antibodies can be produced using the sugar antigen obtained above by administering the antigen to a mammal and collecting antibodies produced in vivo according to a conventional method. There are no particular limitations on the mammal used for antibody production, and examples include rabbits, guinea pigs, mice, sheep, goats, cows, and horses. For production of antibodies, for example, dilute a predetermined amount of the above antigen to an appropriate concentration with physiological saline, mix with this an adjuvant such as Freund's incomplete adjuvant or Freund's complete adjuvant as necessary, and use the resulting suspension. This is done by administering. The above administration is carried out subcutaneously, intramuscularly, intraperitoneally, intravenously, orally, etc., preferably by subcutaneously, intraperitoneally, or intravenously. The number of administrations, dosage, etc. can be appropriately determined according to conventional methods.
For example, the above suspension is injected intradermally into rabbits (antigen amount: 0.05 to 5 mg/dose), and then every two weeks,
Immunization can be achieved by administering the vaccine for 10 months, preferably 1 to 3 months. Antibodies are collected by collecting blood from the immunized animal at a time when antibodies are produced in large quantities after the final administration of the suspension, usually 1 to 2 weeks after the final administration, centrifuging the blood, and separating the serum. This is done by sampling. Further, the above-mentioned serum may be further purified by conventional purification techniques such as salting out, absorption method, and affinity chromatography. The thus purified antibody is α-fucopyranosyl-(1→3)-, -(1→4)- or -(1→6)-
This is an antibody that can specifically recognize galactopyranosyl groups. In particular, in the present invention, 3′-
When using α-L-fucopyranosyl-α-lactose, O-α-L-fucopyranosyl-(1→
3) A highly specific antibody that can recognize -O-β-D-galactopyranosyl group is used as a hapten for 4'-
When using α-L-fucopyranosyl-α-lactose, O-α-L-fucopyranosyl-(1→
4) A specific antibody that can recognize -O-β-D-galactopyranosyl group is also used as a hapten for 6′-α-
When L-fucopyranosyl-α-lactose is used, O-α-L-fucopyranosyl-(1→6)-
Specific antibodies capable of recognizing the O-β-D-galactopyranosyl group can be produced. The antibody produced above binds to cancer cells such as gastrointestinal cancer, such as human colon cancer cells and mouse teratocarcinoma stem cells, but binds to normal tissues such as colon mucosa, liver, gallbladder, pancreas, lung, thyroid, thymus, etc. It has the characteristic that it does not bind to normal human tissues such as lymph nodes, muscles, connective tissues, and blood vessels, and normal mouse tissues such as small intestine, large intestine, liver, kidney, epididymis, and ovary. Furthermore, according to the research of the present inventors, α-fucopyranosyl-(1→3)-, -(1→4)- or -(1→6) −
It was discovered that cancer-related sugar chains containing galactopyranosyl groups are produced and also present in the body fluids of patients. Therefore, α-fucopyranosyl-
By using antibodies that can specifically recognize (1→3)-, -(1→4)- or -(1→6)-galactopyranosyl groups, it is possible to Cancer-related sugar chains can be measured by immunoreaction (antigen-antibody reaction), and cancer can be diagnosed thereby. The present invention also provides a method for measuring cancer-related sugar chains, a method for diagnosing cancer, and a cancer diagnostic kit used in these methods. The antibodies used in the measurement of cancer-related sugar chains and cancer diagnosis of the present invention include the antibodies obtained as described above, ie, α-fucopyranosyl-(1→3)-,
Any antibody that can specifically recognize -(1→4)- or -(1→6)-galactopyranosyl group can be used. Specifically, O-α-L-fucopyranosyl-
(1 → 3) -Antibody that recognizes -O-β-D-galactopyranosyl group (hereinafter referred to as "antibody-"), O-
α-L-fucopyranosyl-(1→4)-O-β-D
-Antibody that recognizes the galactopyranosyl group (hereinafter referred to as "antibody-"), an antibody that recognizes the O-α-L-fucopyranosyl-(1→6)-O-β-D-galactopyranosyl group ( (hereinafter referred to as "antibody-"). Among these, antibodies-
is preferred. Cancer-related sugar chains are α-fucopyranosyl-(1→3)-, -(1→4)- or -(1→
6) - Glycoproteins and/or glycolipids having a galactopyranosyl group can be mentioned. The measurement of cancer-related sugar chains of the present invention is carried out according to a conventional method, for example, specifically as follows. That is, when cells and/or tissue pieces are used as measurement materials, the measurement is carried out according to the usual indirect immunization method. According to this method, the antibodies of the invention are immunoreacted with cells suspended in a buffer such as physiological saline or normal phosphate buffered saline (PBS), or with tissue sections fixed on glass slides. After thoroughly washing the cells or tissue pieces with the above-mentioned buffer, the presence or absence of the antibody of the present invention bound to the cells or tissue pieces may be examined by a conventional labeled antibody method or by using labeled protein A. In the labeled antibody method, a labeled antibody against the antigen of the animal for which the antibody of the present invention was produced, such as labeled anti-rabbit immunoglobulin G antibody, anti-mouse immunoglobulin G antibody, anti-goat immunoglobulin G antibody, etc., is appropriately selected. can be used. As a labeling agent for the labeled antibody and labeled protein A, various fluorescent labeling substances or enzyme labeling substances can be used. Typical fluorescent substances include, for example, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate (XRITC), rhodamine B isothiocyanate, and dichlorotriazine fluorescein. Examples of enzyme labeling substances include peroxidase (POX), microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-
Examples include Nase, P-Nase, and the like. Antibodies or protein A labeled with these
As Acta., either a commercially available one or one prepared according to a conventional method may be used.
Endocrinol.Suppl., 168 , 206 (1972) and Proc.
Nat. Acad. Sci., USA, 57 , 713 (1967)].
In this method, the cells or tissue pieces treated with the antibody of the present invention are reacted with a labeled antibody or labeled protein A that has been diluted in advance with the same buffer as above, and the cells or tissue pieces are sufficiently isolated in the same manner as above. After washing, the labeling activity (fluorescence activity or enzyme activity) present on the cells or tissue pieces is measured according to a conventional method. Conventional methods can also be followed when using a liquid as the measuring material. Here, as the body fluid, for example, blood, cell tissue fluid, lymph fluid, pleural fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, cerebrospinal fluid, saliva, etc. or centrifuged supernatant after solubilizing the above-mentioned cells or tissue pieces can be used. I can do it. The centrifuged supernatant after solubilizing the cells or tissue pieces can be obtained by a conventional method such as a homogenate method or solubilization using a solubilizing agent, followed by centrifugation and collecting the supernatant.
Furthermore, when blood is used, it is usually preferable to use it as serum or plasma. The amount of body fluid used for measurement may be about 0.1 to 10 ml. The method of the present invention uses the various body fluids mentioned above as measurement materials,
Preferably, this is carried out by conventional competitive radioimmunoassay (RIA) or enzyme immunoassay (EIA). The operations, procedures, etc. of these methods can follow conventional methods. That is, in a normal solvent,
A fixed amount of standard antigen, labeled antigen, and antibody are subjected to a competitive reaction, then the antigen-antibody complex (immune complex) and unbound antigen are separated, the labeling activity of either one of them is measured, and the reaction with known concentration of standard antigen is performed. Create a standard curve. Similarly, a test sample (body fluid) of unknown concentration is used instead of the standard antigen to measure its labeling activity, and the amount of immunosensitive substances (cancer-related sugar chains) against the used antibody in the test sample is determined from the standard curve. can be quantified. As the standard antigen, a substance (antigen or its hapten) that is immunosensitive to the antibody used can be used. As the hapten, for example, when an antibody is used, 3'-α-L-fucopyranosyl-α-lactose is used, and when an antibody is used, 4'-α-L-fucopyranosyl-α-
When using lactose and antibodies,
An example is 6'-α-L-fucopyranosyl-α-lactose. Further, as antigens, antigens corresponding to each of the above-mentioned haptens, specifically, the above-mentioned haptens and carrier proteins as obtained in the antigen production examples described later,
For example, a conjugate with PIP-BSA can be exemplified. As the standard antigen, a standard antigen labeled with a radioactive substance such as ¹²⁵ I or ³ H, or the various enzyme labeling substances mentioned above may be used. When labeling a standard antigen by introducing the above-mentioned radioactive iodine, for example, the isothiocyanate body (hapten-isothiocyanate conjugate) described in the production of the antigen or the conjugate thereof with a carrier protein, specifically, the following. 3′-4′- or 6′-α-fucopyranosyl-α as obtained in the production example of the antigen
-Lactose-PIP, or its conjugate with BSA, can be routinely labeled using the Bolton-Hunter reagent [J.Biol.Chem., 254, 9349-9351 (1979)].
In addition, oxidative iodination using chloramine T [Nature, 194 , p. 495 (1962), Biochem.J.89,
114 (1963)] in which an iodized tyrosine group is bonded to the PIP group by the above-mentioned isothiocyanate coupling method, or BSA
It is also possible to use a group in which the tyrosine residue of the group is similarly iodinated. In addition, when introducing ³ H, the standard antigen is labeled by subjecting it to a reduction reaction using, for example, NaB ³ H ₄ or by acetylation with (C ³ H ₃ CO) ₂ O, according to a conventional method. A labeled antigen can be obtained. The solvent for the measurement system is one that does not adversely affect the immune reaction, such as water, physiological saline, 0.1 molar Tris-HCl buffer (PH = 7.5), 0.1 molar phosphate buffer (PH = 7.4), etc. A buffer of 6 to 7.8 is preferred.
The above immune reaction is carried out according to a conventional method at 45°C or lower, preferably 4 to 40°C, for about 1 to 40 hours. The immune complexes generated by the reaction and the unbound antigen can be separated by a known method, for example, after the dextran-activated charcoal method, or by using a second antibody against the antibody, such as a goat antibody when rabbit antibodies are used in the above method. After reacting with rabbit 1gG antibody, etc., it may be separated by centrifugation. Hereinafter, a specific example of the above measurement method will be described in more detail. 5 to 10 μg of 3′-α-L-fucopyranosyl-α-lactose-PIP obtained in the antigen production example described below
is labeled with ¹²⁵ I using Bolton-Hunter reagent (room temperature, approximately 60 seconds) to produce a labeled antigen. 3'-α-L-fucopyranosyl-α-lactose is used as a standard antigen and an antibody is used as an antibody.
0.2 ml of 0.1 M phosphate buffer (PH = ₇ ) containing 0.5% BSA and 0.02% NaN, 0.1 ml of the above labeled antigen (approx.
10,000 cpm), 0.1 ml of antibody at an appropriate concentration, and 0.1 ml of standard antigen at various concentrations are incubated at 4°C for 24 hours. Then 0.1ml normal pig serum and 0.5ml
Add dextran-activated charcoal suspension, leave at 4°C for 30 minutes, and then centrifuge (3000 rpm, 30 minutes), or add 0.1 ml of goat anti-rabbit IgG antibody at an appropriate concentration, incubate at 4°C for 24 hours, and repeat the same procedure. Immune complexes and unbound antigen are separated by centrifugation and their radioactivity is measured. Determine the radioactivity for each concentration of the standard antigen, or determine the binding between the antibody and the labeled peptide when the binding rate (B0) between the standard antigen and the antibody corresponding to the titer of the antibody used is 100%. Determine the percentage of body (B) and create a standard curve. Furthermore, radioactivity or percentage can be determined in the same manner using a sample of unknown concentration in place of the standard antigen, and cancer-related sugar chains in the sample can be quantified from this value using the standard curve. Furthermore, by the above method, it is possible to measure cancer-related sugar chains having a 3'-α-L-fucopyranosyl-α-galactopyranosyl group in body fluids. Furthermore, in the above, the antibody-
4'-α-L-fucopyranosyl-α-galactopyranosyl group or 6'-α-L-
Cancer-related sugar chains containing fucopyranosyl-α-galactopyranosyl groups can be measured. A particularly convenient method for carrying out the above measurement method of the present invention is a method using a kit for determining the amount of cancer-related sugar chains in a liquid such as plasma or serum. Such kits contain antibodies that specifically perform antigen-antibody reactions with cancer-related sugar chains, ie, α-fucopyranosyl-(1→3)-, -(1→4)-, or -(1→6)-.
It is necessary to contain an antibody that can specifically recognize galactopyranosyl groups. Stabilizers and/or preservatives such as glycerol and bovine serum proteins can be added to the antibody reagent.
Preferably, the antibody reagent is frozen and thawed, and the kit can contain a water-soluble or water-miscible solvent. Furthermore, the antibody reagent may be supplemented with a buffer to maintain a constant pH of the reconstituted reagent system and/or a preservative and/or stabilizer to prevent the sample from deteriorating before use. can. Although a buffer solution is not considered to be an essential component of the Kitt reagent, it is preferable to use one that has a pH of 6 to 7.8 when carrying out the measurement method of the present invention. Although the reconstitution agent preferably contains water, part or all of the water may be replaced by a water-miscible solvent. Water-miscible solvents are well known to those skilled in the art, and include, but are of course not limited to, glycerin, alcohols, glycols, glycol ethers, and the like. Thus, according to the present invention, cancer-related sugar chains can be advantageously measured. By comparing the measured cancer-related sugar chain level with that of healthy people,
Carcinoma of the gastrointestinal tract, etc. from early stage to late stage in the subject,
In particular, colon cancer can be diagnosed. Therefore, this method is particularly useful for early detection of cancer. In order to explain the present invention in more detail, examples of producing sugar antigens (fucose antigens) and antibodies will be given below. Production example of antigen 1 (1) Production of 3'-α-L-fucopyranosyl-α-lactose-phenethylamine derivative 0.1 mmol of 3'-α-L-fucopyranosyl-α-lactose and β-(p-aminophenyl)
Place 3.5 mmol of ethylamine in a sealed container.
The reaction was stirred at room temperature for 15 hours. 0.5 ml of pure ethanol was added to the reaction mixture, followed by 1 ml of pure ethanol in which 12 mg of sodium borohydride was suspended, and the mixture was stirred at room temperature for 5 hours. Then water 4
ml to dilute, and add glacial acetic acid dropwise under ice cooling.
Adjusted to PH5.6. After distilling off the ethanol under reduced pressure, water was added to make 5 ml of the reaction mixture, which was passed through a Sephadex G-10 column (2.5 x 100 cm).
Elution was performed with 1M acetic acid-pyridine buffer (PH=5.0). The eluate was fractionated into 5 ml portions, and each fraction was subjected to measurement of neutral sugars by phenol-sulfuric acid reaction.
Absorbance was measured at OD ₂₈₅ nm, and fractions with matching peaks were collected and lyophilized. Frozen samples were soaked in 2mM acetate-pyridine buffer (PH
5.0) and Watmann CM52 column (0.5
x 20 cm) and unreacted raw material (3′-
After elution of α-L-fucopyranosyl-α-lactose), it was eluted with 0.1N aqueous ammonia. Fractionate the eluate into 20 drops (approximately 0.6 ml) and measure neutral sugar and OD ₂₈₅ n in the same manner as above for each fraction.
Absorbance was measured at m, and fractions with matching peaks were collected and freeze-dried. Thus 3′-α-L-fucopyranosyl-α-
A lactose-phenethylamine derivative was obtained.
The sugar composition of this product was determined by gas chromatography [Biochem.Biophys.Acta., 222 , 339-347] and high performance liquid chromatography [Developmental Biology, 90 , 441-444].
(1982)]. (2) 3′-α-L-fucopyranosyl-α-lactose-p-isothiocyanate-phenethylamine derivative (3′-α-L-fucopyranosyl-α-
Production of lactose-PIP) 3′-α-L-fucopyranosyl-obtained in (1) above
α-lactose-phenethylamine derivative
Thiophosgene 65μmol was dissolved in 2ml of 0.1M sodium bicarbonate aqueous solution (PH=8.0).
The mixture was layered on 2.5 ml of chloroform containing mole and stirred vigorously for 1 hour. The reaction mixture was transferred to a centrifuge tube and extracted twice with 2 ml of chloroform to remove excess thiophosgene, the aqueous layer was collected, and nitrogen gas was passed through to remove residual chloroform. Thus 3′-α-L-fucopyranosyl-α-
A lactose-p-isothiocyanate-phenethylamine derivative was obtained as an aqueous liquid. (3) Sugar antigen (3'-α-L-fucopyranosyl-α-lactose-PIP- Production of BSA) The aqueous solution obtained in (1) above was mixed with 0.5M sodium chloride containing 0.2 μmol of bovine serum albumin (BSA).
The mixture was added to a 0.1M aqueous sodium hydrogen carbonate solution (PH=9.5) and stirred at room temperature for 18 hours to react. The reaction mixture was dialyzed against Dulbetskow-treated PBS(-) (physiological saline-phosphate buffer) 21 to remove unreacted 3′-α-L-fucopyranosyl-α.
-Lactose-p-isothiocyanate-phenethylamine derivative was removed. After exchanging the dialysate three times every 12 hours, the dialyzed solution was subjected to the Lowry method and phenol-sulfuric acid reaction, and the amount of protein and neutral sugars were determined. The resulting sugar antigen had approximately 20 moles of 3'-α-L-fucopyranosyl sugar chain bound to 1 mole of bovine serum albumin (BSA). In this way, the desired sugar antigen solution was obtained. This was stored frozen (this will be referred to as "antigen"). Antigen Production Example 2 In the above Antigen Production Example 1, 4'-α-lactose was used instead of 3'-α-L-fucopyranosyl-α-lactose.
A target sugar antigen solution was similarly obtained using L-fucopyranosyl-α-lactose. This was stored frozen (this will be referred to as "antigen"). This sugar antigen is bovine serum albumin (BSA1)
Approximately 25 moles of 4'-α-L-fucopyranosyl sugar chains were bound per mole. Antigen Production Example 3 In the above antigen production example 1, 6'-α-lactose was used instead of 3'-α-L-fucopyranosyl-α-lactose.
A target sugar antigen solution was similarly obtained using L-fucopyranosyl-α-lactose. This was stored frozen (this will be referred to as "antigen-"). This sugar antigen is bovine serum albumin (BSA1)
Approximately 23 moles of 6'-α-L-fucopyranosyl sugar chains were bound per mole. Antibody Production Example 1 1 ml of Freund's complete supplement containing 0.4 mg of the antigen obtained in Antigen Production Example 1 was injected into the footpads of a New Zealand white rabbit. After 3 weeks, the same amount of antigen-containing Freund's complete supplement was injected, and this procedure was repeated every 2 weeks 3 times.
Repeated times. Ten days after the third (final) injection, blood was collected from the test animals and centrifuged to collect antiserum to obtain the antibody of interest. This is called “antibody-
”. Antibodies are stored at -70°C. In addition, the antiserum obtained above was lyophilized to produce antibodies.
A dried product was obtained. Antibody Production Example 2 Using the antigen obtained in Antigen Production Example 2, prepare the desired antibody (antiserum) in the same manner as in Antibody Production Example 1.
I got it. This is referred to as "antibody-". Antibody Production Example 3 Using the antigen obtained in Antigen Production Example 3, prepare the desired antibody (antiserum) in the same manner as in Antibody Production Example 1.
I got it. This is referred to as "antibody-". Examples of antibody specificity tests will be described in detail below. <Antibody specificity test> (1) Centrifuge various cells (500 x g) and dilute with 50 times the volume of phosphate buffered saline (containing calcium and magnesium ions, PH = 7.2).
Wash twice. The obtained cells were suspended in the above-mentioned phosphate buffered saline at a concentration of 1% (V/V), and 50μ of this suspension was added with the antibodies obtained in Antibody Production Examples 1 to 3 (antibody- ~-) previously diluted 20 times in volume with phosphate buffered saline (containing calcium and magnesium ions) or similarly diluted normal rabbit serum as a control. Incubate for 1 hour at ℃. Each cell was then washed with 100 times the volume of phosphate-buffered saline (containing calcium and magnesium ions, PH = 7.2), and then FITC-shared sheep anti-rabbit IgG [Miles-Yeda] Incubate at 4°C for 1 hour using a 1/10 diluted solution (manufactured by J.D. Co., Ltd.) at 4°C. Subsequently, after washing twice with 100 times the volume of the above phosphate buffered saline (PH = 7.2), each cell was examined under an epifluorescence microscope (Olympus model BH-
RFL-LB, manufactured by Olympus Optical Co., Ltd.).
Shoot with Fuji Color Film ASA100 (manufactured by Fujifilm). (2) Rapidly freeze cancer or normal tissue and obtain ultrathin sections using a cryostat (manufactured by American Optical). This was fixed on a glass slide with acetone for 1 minute as a specimen, and then FITC-
FITC-Sheep anti-rabbit IgG-F instead of sheep anti-rabbit IgG
(ab)' ₂ (manufactured by Cappel) and tested in the same manner as above. In (1) and (2) above, the results of examining the reactivity of each cell or tissue piece used with the antibodies are shown in Table 1 below for each antibody. The evaluation symbols for each reactivity in Table 1 indicate the following. +...Stained image is observed. -...No stained image is observed.

【table】

【table】

[Table] In the case of normal rabbit serum used as a control instead of antibodies - to - in the above, no staining images were observed. (3) Using a human colon adenocarcinoma (surgical piece) in which a stained image was observed in the test in (2) above as a specimen,
0.2M3'-α during the first reaction.
-L-fucopyranosyl-α-lactose,
0.2M lactose, 0.2M fucose or 10mg/ml
The test was conducted in the same manner as in (2) above in the presence of BSA. As a result, the stained image was attenuated by 3'-α-L-fucopyranosyl-α-lactose, but
No change was observed in the staining images for lactose, fucose, and BSA. <Antibody specificity test> The specificity of the antibody was investigated using the Ouchterlony double diffusion analysis method as follows. Namely, 1% agar gel (2% Triton
Agar gel containing % _NaN3 ) was stacked on a slide glass, the antibody was placed in the center, and 20 μg of 3'-α-L-fucopyranosyl-
α-lactose-PIP-BSA, 4′-α-L-fucopyranosyl-α-lactose-PIP-BSA,
An aqueous solution containing 6'-α-L-fucopyranosyl-α-lactose-PIP-BSA, α-lactose-PIP-BSA, and BSA was placed and a diffusion test was conducted. The results are shown in FIGS. 1 to 3. FIG. 1 is a diagram showing the state of antibody diffusion, FIG. 2 is a diagram showing the state of antibody diffusion, and FIG. 3 is a diagram showing the state of antibody diffusion. In each figure, (a) is 3′-α-L-fucopyranosyl-α-lactose-PIP-BSA, (b) is 4′-α-L-fucopyranosyl-α-lactose-PIP-BSA, (c)
is 6′-α-L-fucopyranosyl-α-lactose-PIP-BSA, (d) is α-lactose-PIP-
BSA and (e) indicate BSA, respectively. The following can be seen from each figure. That is, the antibody is 3′-α-
L-fucopyranosyl-α-lactose-PIP-
It forms precipitation lines with BSA, but not with other antigens. Antibody- is 4'-α-L
-Fucopyranosyl-α-lactose-PIP-
It forms precipitation lines with BSA, but not with other antigens. Antibody- is 6'-α-L
-Fucopyranosyl-α-lactose-PIP-
It forms precipitation lines with BSA, but not with other antigens. In the above test, the antibody was prepared using 1 ml of the antibody obtained in the above antibody production example.
0.4 mg of BSA was added per tube, left overnight at 4° C., centrifuged, and the supernatant was collected. After removing the anti-BSA antibody, it was used in the above test.

[Brief explanation of the drawing]

FIGS. 1 to 3 are diagrams showing the state of diffusion of the antibodies of the present invention by double diffusion analysis.

Claims (1)

[Claims] 1 α-fucopyranosyl-(1→3)-, -(1→
4) A cancer-related saccharide antigen consisting of a complex of an oligosaccharide containing a - or -(1→6)-galactopyranosyl group and a carrier protein.