JPS5959200A - Measurement of carbohydrate acceptor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for determining carbohydrate receptors in biological samples. In particular, the present invention relates to a method for measuring specific carbohydrate receptors useful in the diagnosis and/or treatment of cancer and other chronic diseases.

It has been reported that many patients diagnosed with cancer have high serum glycoprotein concentrations. This increase in glycoprotein concentration may be due to increased shedding and/or secretion of glycoproteins by malignant cells or increased host glycoprotein synthesis in response to the tumor.

In fact, the increase in serum glycoprotein concentration is small, and since serum glycoprotein varies from person to person and changes over time even in the same person, this is the case. Efforts to measure accurately have been relatively unsuccessful.

Carbohydrate receptors are derived from tumors, ie, from tumor glycoproteins resulting from cell necrosis, or from host tissues resulting from tissue destruction, ie, as a result of tumor growth. Tumor growth induces the migration of host macrophages and neutrophil cells near the tumor. When host cells are brought into contact with tumor cells, degradative enzymes present in the host cell lyso-sone are released. For example, lysosum contains sialidase and β, which can produce glycoproteins with an N-acetylglucosamine moiety at the end.
- Contains various glycosidases including galactosidase. −.

Other lysone malglycosidases, such as N-acetylglucosaminidase, in turn cleave all oligosaccharide groups of glycoproteins to generate other terminal carbohydrates. Each terminal carbohydrate is capable of serving as an acceptor for the appropriate sugar derivative and glycosyltransferase. A steady increase in the amount of this partially degraded glycoprotein can be expected in samples found from patients with cancer or other chronic diseases due to the continuous production of this glycoprotein in the serum of such patients. The increase in the amount of partially degraded glycoproteins may be due to insufficient removal of the degraded glycoproteins during the process.

For example, glycoproteins with newly exposed mannose residues will be slower to modify or remove than the corresponding galactose-terminated glycoproteins. An additional source of glycoproteins with incomplete oligosaccharides may be due to underdeveloped glycosylations of tumor cell glycoproteins. An increase in the amount of incomplete glycoproteins in the serum may result from the destruction of tumor cells by an ineffective system of glycosyltransferases within the tumor cells due to host reaction and/or tumorigenic metastasis. The host also responds to the presence of tumors by producing blood glycoproteins with sugar receptor sites.

Although many specific glycoproteins that are characteristically found in cancer patients have been identified, there is no rapid method for quantitatively measuring carbohydrate receptor classes with oligosaccharide moieties in Plugia. U.S. Patent No. 4,1
Davidson et al. 4.2 to 4.6 in No. 46,608.
describes a tumor-specific glycoprotein that has an isoelectric point and is soluble in perchloric acid. The presence of the tumor-specific glycoprotein is detected by mixing the serum sample with perchloric acid to partially precipitate the tumor-specific glycoprotein, and then using the perchloric acid-soluble portion by gel electrophoresis or isoelectric focusing. be. Isselpachiel et al., in US Pat. No. 4,261,976, describe glycopeptides that inhibit the growth of malignant cells or tumors. Glycopeptides obtained from animals or humans with malignant cells or tumors are soluble in phosphotungstic acid, have a molecular weight of approximately 3600, and are substrates for isoenzyme of serum galactosyltransferase (GT-T Oyohi GT-41). It is. The presence of glycoseptide can be determined by determining a known amount of galactosyltransferase'k (ti11) in a sample containing it. Plotkin et al. describe an enzymatic non-aggressive method for the detection of cancer in mammalian tissues that consists of culturing urine containing cells scraped from the animal tissue and measuring the galactosyltransferase activity of the cells. Plotkin et al. [0 ，
80102296 describes a method for determining whether a mammalian tissue cell is malignant by testing cells from the mammal to determine whether the glycoproteins are significantly altered compared to non-malignant cells.

Glycoproteins can be measured indirectly by adding to the cells a landmark that has a specific affinity for galactose or galactose residues and can be detected by non-chemical methods, ie a lectin, and then detecting the amount of the landmark bound to the cell.

It is an object of the present invention to provide a method for directly detecting and quantitatively determining the amount of both sets of carbohydrate receptors, especially glycoproteins with common oligosaccharide moieties, in biological samples.

The present invention comprises: 9-(α) a sample containing a carbohydrate receptor to be measured; and (1) a sugar-nucleic acid acid that allows the sugar moiety of the donor to specifically bind to the carbohydrate acceptor to be measured; (11) a source of enzymes capable of effecting a reaction between the carbohydrate acceptor and the donor; and effective to produce an amount of carbohydrate acceptor-sugar conjugate proportional to the amount of carbohydrate acceptor present in the sample; (b) measuring the amount of carbohydrate-sugar conjugate as a measure of the amount of carbohydrate acceptor present in the sample; Concerning a method for measuring carbohydrate receptors.

The methods of the invention are particularly relevant to the measurement of carbohydrate receptors, particularly glycoproteins useful in the diagnosis and treatment of cancer and other chronic diseases.

The invention further provides that in the presence of manganous chloride it reacts with uridine 5'-diphosphate galactose (UDP-galactose) and adenosine 5'-triphosphate, is insoluble in perchloric acid and phosphotungstic acid, and is insoluble in dodecyl sulfate. Approximately 60,000-80,0 as determined by polyacrylamide gel electrophoresis using sodium
The present invention relates to galactose receptor protein (GAP), which is characterized as a receptor protein capable of forming a complex with a subunit molecular weight of 0.00 Daltons. Measurement of this galactose receptor protein is useful in the diagnosis and/or treatment of cancer and other chronic diseases.

"Carbohydrate receptor" refers to a carbohydrate acceptor that has a recognition site for glycosyltransferase and is specific for measuring carbohydrate receptors and sugar-nucleic acid group combinations. A type of molecule that accepts a sugar moiety. Carbohydrate receptors measured by the method of the invention include glycoproteins, glycolipids, glycopeptides and polysaccharides. Of particular interest are glycoproteins with terminal carbohydrate moieties that exhibit incomplete glycosylation. Included within the scope of ``1 carbohydrate acceptor'' are the terminal carbohydrate moieties on the oligosaccharide continuum of N-linked and 0-linked glycoproteins. Incomplete oligosaccharide buttons are linked to specific enzymes of altered glycoproteins. Target or chemical tagging is possible.

According to one particular embodiment of the method of the invention, the mixture of serum sample, labeling source and enzyme source is incubated for a period of time sufficient to produce a detectable amount of carbohydrate receptor-glycoconjugate. The enzyme-catalyzed reaction is completed and the amount of conjugate produced is measured as a measure of the carbohydrate acceptor in the sample. The reaction time required to form a detectable carbohydrate receptor-sugar conjugate is not critical. Reaction times will vary depending on, for example, carbohydrate receptor concentration, reagent concentration, etc. in the sample. Also, those with knowledge in this technical field will be able to easily confirm this. The method of the present invention is illustrated by the following reaction scheme.

aA+5rya-yvc! N5cA+jsva+Nv
c However, CA represents a carbohydrate acceptor, BUG-NUC represents a donor consisting of a sugar-nucleobase combination, ENZ represents a carbohydrate acceptor and a specific enzyme for the donor, CA,
SUG stands for the carbohydrate receptor-sugar complex produced and NUC stands for the released nucleic acid.

The donor substrate used in the method of the invention consists of a vine sugar-nucleic acid group association specifically recognized by the carbohydrate receptor being measured. That is, the carbohydrate acceptor can accept the sugar moiety of the sugar-nucleic acid group association in the presence of an enzyme and, if necessary, a substrate converting agent. For example, in the measurement of serum glycoproteins with terminal N-acetylglucosamine residues, uridine 5'-diphosphonate galactose (UDP-galactose) is
-Used as a feeder for the production of acetylglucosamine-galactose complexes. The donor can be tagged with various tags that allow it to produce easily detectable carbohydrate acceptor-glycoconjugates. - tags, fluorescent tags, or radioisotope tags. Preferably, a radiolabeled supply is used. This radiolabeled supply may include, for example, tritium (3H), carbon-14C" Various radioactive tags can be attached. If a radiolabeled supply body is used in the method of the present invention, tritium (3H) is used as a radioactive tag.
) is recommended. The specific feedstock used will be readily ascertainable to those of ordinary skill in the art by the carbohydrate acceptor being measured, labeled or untagged. Convenient supplies for the present invention are either commercially available, labeled or unlabeled, or readily synthesized by conventional methods.

The amount of donor used will vary depending on the particular activity of the system, ie, the particular carbohydrate receptor being measured and the particular enzyme used, but can be readily ascertained by one of ordinary skill in the art.

As mentioned above, the method of the invention requires 14-enzymatic transfer of a sugar moiety to a carbohydrate receptor. The enzyme source used in the method of the invention specifically facilitates the reaction of the carbohydrate acceptor and donor. The particular enzyme source used will be determined by the carbohydrate receptor being tested and the particular source used. Enzyme sources, including enzymes, isoenzymes and variants, are known in the art, and the particular enzyme source required can be readily obtained by one of ordinary skill in the art depending on the particular carbohydrate acceptor delivery system used. For example, in testing serum glycoproteins with terminal N-acetylglycosamine residues using UDP-galactose as the donor, galactosyltransferase is used as the enzyme. Also, some enzymes require the presence of cofactors to increase enzyme activity. These cofactors are well known in the art, and the particular cofactor used will be readily ascertainable to those of ordinary skill in the art. For example, if galactosyltransferase is used as the enzyme source, manganous chloride is used as the cofactor. The enzyme source concentration used will vary depending on the particular activity of the system, ie, the carbohydrate acceptor being measured, the particular donor, and the enzyme used, but will be readily ascertained by one of ordinary skill in the art. Importantly, however, although the serum sample to be analyzed contains a certain amount of enzyme, the method of the present invention generally requires the addition of an exogenous enzyme source in an amount sufficiently in excess of the amount of enzyme normally found in serum. In other words, carbohydrate receptors are limiting reagents. The enzyme and donor concentrations are therefore adjusted so that the amount of carbohydrate-sugar combination produced is proportional to the carbohydrate acceptor concentration present and is not limited by the concentration of either donor or enzyme.

Still further, for the feed and enzyme sources, the method of the present invention requires the presence of a substrate converting agent for detectable carbohydrate glycoconjugate formation. Depending on the specific feed strength used in the system and the feed concentration, a substrate converting agent may be necessary to prevent degradation of the feeder, thereby causing the sugar moiety of the sugar-nucleic acid group to specifically bind to the carbohydrate receptor of interest. Substrate converting agents may be necessary when donor concentrations are low or when the particular donor used has bonds that are susceptible to cleavage by serum enzymes. Substrate conversion agents are well known in the art, and the particular substrate conversion agent required will be readily ascertained by one of ordinary skill in the art depending on the particular feedstock used and carbohydrate acceptor measured. For example, in the present invention, serum glycoproteins with terminal N-acetyldaricosamine residues are analyzed using adenosine 5'-tribosphate (A
TP).

The temperature used in the method of the invention is generally the temperature that is optimal for the enzyme activity of the particular system. Temperature generally varies depending on the particular enzyme source.

Further, the temperature is preferably kept essentially constant at ±5°C, preferably ±2°C of the optimum temperature.

The amount of carbohydrate acceptor=glycoconjugate-17= compound obtained as a result of the enzyme-catalyzed reaction is determined as a measure of the carbohydrate acceptor present in the sample. The amount of specific carbohydrate receptors measured in samples obtained from patients with or suspected of having cancer or other chronic diseases may be used in normal samples obtained from healthy subjects as a means of determining the amount of carbohydrate receptors associated with specific diseases. compared to the amount of carbohydrate receptors found in

The carbohydrate receptor-sugar conjugates produced can be directly measured using tagged feeders. Using a tagged feed, such as a feed containing a radiolabeled sugar moiety, a radiolabeled carbohydrate receptor-sugar conjugate is produced and measured using conventional radiochemical analysis techniques. If a housing-tagged donor is used, the enzymatic contact reaction between the carbohydrate acceptor and the donor is generated and completed before the amount of the original carbohydrate acceptor-glycoconjugate is measured. Methods for terminating this reaction are well known in the art and include, for example, large excess addition of unlabeled feedstock, heat treatment, i.e., increased temperature to inactivate the enzyme, restriction of the cofactor in the case of cofactor use. etc., the enzyme source becomes non-active 1 < 1. If a radioactively labeled donor is used, one additional unlabeled donor can be added to terminate the catalytic reaction. It is also necessary to separate the tagged carbohydrate acceptor-sugar conjugate produced from the unreacted porous feed prior to determining the amount of tagged carbohydrate acceptor-sugar conjugate produced. For example, when the carbohydrate receptor to be measured is m-protein, the carbohydrate receptor-sugar complex can be separated by precipitation in phosphotungstic acid. Similarly, if a glycolipid is to be measured, the corresponding carbohydrate receptor-sugar complex produced can be separated using a lipid extraction method.

Carbohydrate receptor produced if unlabeled donor was used -
The amount of glycoconjugate can be measured indirectly by measuring the amount of nucleic acid bases released by the reaction between the carbohydrate acceptor and donor. For example, if UDP-galactose is used as the donor, UDP will be released upon reaction with carbohydrates in the sample. Therefore, the generated U
The amount of DP can be measured by enzymatically converting the released UDP nucleic acid salts into products that can later be reduced to form reduced nicotinamide adenine dinucleic acid salts (NADII). The NADII thus produced is a measure of the carbohydrate receptor-sugar conjugate produced.

Importantly, the concentrations of the donor and enzyme source (endogenous and/or exogenous) do not require precision. Effective amounts of donor and enzyme source are added such that sufficient sugar is transferred from the donor to the carbohydrate acceptor to produce an amount of carbohydrate acceptor-sugar conjugate in proportion to the amount of carbohydrate acceptor in the sample. Effective amounts of the donor and enzyme source are readily ascertainable to those of ordinary skill in the art. The effective amount of donor is at least equal to, and preferably greater than, the amount of carbohydrate acceptor in the sample. The effective amount of enzyme is at least equal to, and preferably greater than, the amount of enzyme required to complete the transfer of sugar from donor to carbohydrate acceptor).

Importantly, due to the fact that excess enzyme is added during the procedure of the analysis itself, the sample being analyzed generally does not require the special handling precautions required for enzymatic methods. Therefore, denaturation of the endogenous enzymes due to high temperatures, such as due to sample incubation, time, freeze-thaw, etc., is not important and does not affect the analytical results.

In addition to the analytical sample types for carbohydrate receptors, there are biological samples such as body fluids, tissue samples and serum. Serum samples are preferred for increased sensitivity and specificity as well as for ease of manipulation.

Table 1 shows various sources, enzymes, and substrate converting agents that can be used to measure specific carbohydrate receptors according to the method of the present invention.

The following symbols are used in Table 1: UDP-uridine 5'-diphosphate, Cf1P-cytidine monophosphate, Gl)P-guanosine diphosphate, =21- ATP-adenosine 5'-triphosphate, AMP-
Adenosine monophosphate, CTP-Cytidine triphosphate table ■ Receptor protein -Sulfurase Charyl acid receptor CMP-Sha4 Charyl trans AM
P protein ferase fucosyl receptor GDP-7 course fucosyl trans CTP
If a supply body with a tag is used, tritium (3H) tags, ie, 22-UDP-CH)-galactose, etc. can be used.

Table I shows various analytical formats that can be used with the method of the invention. Many other methods could be developed, as will be apparent to those of ordinary skill in the art. As is known in the art, each carbohydrate receptor has a specific glycoside linkage through which the sugar moiety can be transferred. To further increase degradation and specificity, a specific set of enzymes may be used to interface the transfer of the sugar moiety to the carbohydrate acceptor and create a specific molecular bond. For example, in the analysis of serum glycoproteins with terminal N-acetylglucosamine residues using UDP-galactose as the donor, both sets of galactosyltransferases transfer the galactose into the β1→4 linkage. Different sets of galactosyltransferases can be used to transfer the binding.

The specific combination of enzymes required for each enzyme depends on the desired molecular bonding, which is readily ascertainable to those familiar with the field. The invention will be described in further detail by the following examples.

Example I Serum sample 80μe on microtitrator plate QH) UDP-galactose 0.621tci, 10 mA/ATP
(pH6,8) 10.0fill, 0.1 M M
nC (3,10,011B, 0.1M sodium forcecodylate (pH 7,4) 30.0μE1 Milk galactosyltransferase (100μg Pro t/m
l, 0.45 units/rrtl; 1 unit contacts the transfer of 1 μM galactose per minute at 30°C)10.
0. u/? , and 1000 pM/pe untagged UD
60 μβ of a solution containing 4.0 μE of P-galactose was added. The microtitrator plate was mounted with acetite plate mounting medium prepared in advance. The reaction mixture was incubated in an 87°C water bath for 60 minutes.

After culturing, the acetite plate cover was removed and the plate was placed on wet water.

The reaction mixture was supplemented with plain UDP-galactose (10rnM
) 10μe was added. The resulting mixture containing the radiotagged GAP-galactose complex was applied to glass fiber paper (Whatman A984-A, H, glass microfiber filter).

The glass fiber paper was dried in an 80°C oven for 8-10 minutes and placed on lacrimal paper pre-moistened with 5% phosphotungstic acid solution in 02NHCp. After 5 minutes, apply 0.2 N to the glass fiber paper.
It was placed in a solution containing 5-thiphosphotungstic acid in HCe. After 155 minutes, the glass fibers were removed from the phosphotungstic acid solution and the water was blotted between paper towels at 0.1N.
Transferred to HC1j solution. After 10 minutes, the paper was removed from the Hce solution, blotted with a paper towel, and transferred to a second 0ANHCβ solution. After another 100 minutes, the glass fiber was taken out from the HCe solution, drained with a paper towel, and transferred to an absolute ethanol solution. 5
After a few minutes, the glass fiber paper was removed from the ethanol and blotted with a paper towel and transferred to a second ethanol solution. After a further 5 minutes, the glass fiber paper was taken out from the ethanol solution, blotted between paper towels, and dried in an oven at 25°C and 980°C for 10 minutes. G with radioactive tag
The glass fiber paper containing the AP-galactose complex was placed in a scintillation hing and added with lOd of scintillation solution (
INSTA-GEL) and the tagged formulation (3H) precipitated onto glass fiber paper was measured in a TRI-CARB liquid scintillation counter for 1-2 minutes.

Example 11 The galactose receptor protein-galactose complex (GAP-galactose) produced is characterized by the following properties: 1) Most of the serum radiotagged GAP-galactose complex is 0.1% sodium dodecyl sulfate. approximately 60,000-80 as determined by autofluorography after using polyacrylamide gel electrophoresis in the presence of
,000; 2) More than 50% of the total GAP-galactose complex has a subunit molecular weight of 50
%26- precipitated using saturated ammonium sulfate solution; 8)
GAP-galactose complex is insoluble in 5% phosphotungstic acid solution; 4) More than 90% of GAP-galactose complex is insoluble in 0.6M perchloric acid solution at 0°C.

As mentioned above, the method of the present invention is convenient for the analysis of carbohydrate receptors, and also provides a convenient method for the diagnosis and/or treatment of cancer and other chronic diseases. Method and Examples of Example I■
Serum samples obtained from patients previously diagnosed with various cancers such as colon, breast, lung, and pancreatic cancer as well as other chronic diseases such as Hodgkin's disease using galactose receptor proteins with characteristics of 150 out of 239 (64.4%
) have been found to be at increasing levels. The method for measuring carbohydrate receptors, especially galactose receptor proteins having the characteristics of Example 2, by the method of the present invention is useful for the diagnosis and treatment of cancer and other chronic diseases.
A) Convenient in combination with other assays for cancer-landmarks, such as assays for cancer.

A person knowledgeable in this technical field will recognize many equivalents to the specific MtplIJ described in the specification of the present invention, or will be able to ascertain them without routine experimentation. These equivalents are also considered to be within the scope of the following claims.

Patent applicant: Abbott Laboratories
People Patent Attorney Ryoji Kawase 1st and 4th,
;Het-Al Agent Patent Attorney Takehiko Saito, / ,\2
1.

Procedural amendment c method) % formula % 1. Indication of the case 1982 J.P.A. No. 137878 2. Name of the invention Carbohydrate receptor measurement method 3. Person making the amendment Relationship with the case Patent applicant name Abbott Laboratories Akasaka Taisei Bill (telephone: 582-7161): I typed and typed the hand-written detailed statement as shown in the appendix with frosted handwriting on the application. However, there are no amendments to the content.

Claims (1)

[Scope of Claims] Lα) A sample containing the carbohydrate receptor to be measured is supplied to i) a donor comprising a sugar-nucleic acid combination having a sugar moiety capable of specifically binding to the carbohydrate receptor to be measured; and ii) a carbohydrate and After mixing the donor reaction with the accessible enzyme source and adding an amount of the donor and enzyme source effective to produce an amount of carbohydrate acceptor-glycoconjugate proportional to the amount of carbohydrate acceptor present in the sample, b ) A method for measuring carbohydrate receptors in a sample, which comprises measuring the amount of carbohydrate receptor-sugar complexes produced using the amount of carbohydrate receptors in the sample as a measure. 2. The method according to claim 1, wherein a substrate converting agent capable of specifically binding the sugar moiety to the carbohydrate receptor while preventing the alteration of the sugar-nucleic acid salt is mixed with the sample, the supply body, and the enzyme. a. The method according to claim 1, wherein a radiolabeled sugar-nucleic acid complex is used as a supply body. 4. The method according to claim 1, wherein the sample is a serum sample. 5. The method according to claim 1, wherein a dynamic factor is added to the enzyme source. 6α) Contacting a sample containing a carbohydrate receptor to be measured with 1) a supply body consisting of a sugar-nucleic acid tolerant complex having a sugar moiety that can specifically bind to the carbohydrate receptor to be measured; 11) a reaction between the carbohydrate receptor and the supply body; and 111) carbohydrate receptors that prevent denaturation of sugar-nucleic acid salts and specifically bind sugar moieties to carbohydrate receptors when mixed with a substrate converting agent and proportional to the amount of carbohydrate receptors present in the sample. adding an effective amount of a donor and an enzyme source to produce a carbohydrate conjugate amount to produce a carbohydrate acceptor-viscous conjugate; b
) After completing the reaction between the carbohydrate acceptor and the donor, C) measuring the amount of the carbohydrate acceptor-viscous conjugate produced using the amount of carbohydrate present in the sample as a measure. A method for measuring carbohydrate receptors in 7. The method according to claim 6, wherein the sample is a serum sample. 8. The method according to claim 6, wherein a radioactively labeled sugar-nucleic acid complex is used as a supplier. 9. The method according to claim 6, wherein a cofactor is added to the enzyme source. 10. Reacts with UDP-galactose, galactosyltransferase and adenosine 5'-triphosphate in the presence of manganous chloride and is insoluble in perchloric acid and phosphotungsten, phosphoric acid and electrolytic polyacrylamide gel using sodium dodecyl sulfate. A galactose receptor protein characterized by a receptor protein capable of forming a complex with a molecular weight of 60.0 (1 O-80,000) as determined by electrophoresis. 11, α) UDP- (
After mixing sH)-galactose, galactosyltransferase and adenosine 5'-triphosphate to form a galactose receptor protein-(3H)-galactose complex, b) determining the galactose receptor protein using the galactose receptor protein present in the sample as a measure. 11. A method for measuring galactose receptor protein in a sample, which comprises measuring the amount of protein-(3H)-galactose complex. 12. The method according to claim 11, wherein the sample is a serum sample.