KR101391319B1 - Antibody-Lectin Sandwich Assay for the Measurement of Breast Cancer Marker - Google Patents

Abstract

The present invention relates to a method for measuring antibody-lectin sandwich for the measurement of breast cancer markers and a method for screening for breast cancer.
In the method of measuring cancer marker using the antibody and lectin of the present invention, various kinds of antigens can be assayed regardless of the kind of O-linked glycan exposed to the antigen, and the method of measuring the marker using the antibody / It can be used as a substitute for enzyme linked immunoassay based on the result of measurement corresponding to binding immunoassay.

Description

(Antibody-Lectin Sandwich Assay for Measurement of Breast Cancer Marker)

The present invention relates to a method for measuring a breast cancer marker comprising an O-linked glycan using an antibody and lectin.

Enzyme Linked Immunosorption Assay (ELISA) is currently being used in hospitals and laboratories for the analysis of diseases. This method generally requires two types of monoclonal antibodies that recognize different recognition sites of the antigen. Antibodies specifically recognizing and binding to the antigen are produced through a separate genetic engineering process. Depending on the type of disease marker, only one type of monoclonal antibody that recognizes an antigen has many limitations in detecting the antigen. Further, since the intensity of color developed depending on the enzyme reaction is measured according to the concentration of the antigen, a separate optical measuring instrument is required. These methods are complex and time-consuming. Therefore, a detection system capable of rapid measurement rather than a time-dependent optical signal due to an enzyme reaction is required.

Conventional enzyme-linked immunosorbent assays require two types of monoclonal antibodies against one type of marker. In order to use a marker specific antibody, a separate preparation is required. This takes a lot of time and expense, and verification of antigen affinity of each of the produced monoclonal antibody pairs is required. However, lectins are not required to be produced separately, and they are economical compared with antibodies because they are mainly extracted from plants. Lectins have the characteristic of specifically recognizing and binding specific saccharides and their binding ability is already verified, so that it is possible to measure and quantitate cancer markers including sugar chains. Therefore, if a cancer marker contains a specific sugar chain and a monoclonal antibody capable of recognizing a cancer marker is present, detection of a cancer marker corresponding to an enzyme-linked immunosorbent method using two monoclonal antibodies with only one monoclonal antibody It is possible, but it is not yet a specific invention.

Accordingly, the present invention aims to establish a measurement method capable of measuring various antigens including cancer markers using antibodies and lectins based on the characteristics of the lectins. In particular, as cancer progresses, cancer cells are exposed to O-linked glycans, which are different from normal cells. In normal cells, an O-linked glycan chain is formed in which cell surface ends are exposed to galactose, N-acetylglucosamine, and fructose. In cancer cells, however, immature short-form O-linked glycan chains are formed, The TF antigen or sialic acid to which N-acetylgalactosamine and galactose are linked is exposed. Currently, there is no antibody specifically recognizing only O-linked glycans in cancer cells. Our team has introduced lectins that specifically bind to O-linked glycans to overcome the limitations of these antibodies and to find ways to specifically screen for cancer markers.

The present inventors have sought to find new biomarkers capable of rapidly and accurately diagnosing breast cancer. As a result, the present inventors have completed the present invention by identifying that the biomarkers uncovered are capable of diagnosing breast cancer and determining the prognosis.

It is therefore an object of the present invention to provide a method for detecting a breast cancer marker in order to provide information necessary for diagnosis or prognosis of breast cancer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention,

(i) contacting an antigen that specifically binds to an antibody immobilized on a support with a lectin attached with an optical label substance recognizing an O-linked glycan of the antigen; And

(ii) measuring the amount of lectin to which the optical label substance in contact with the antigen is attached

Lectin < / RTI > sandwich for the measurement of breast cancer markers comprising the antibody-lectin sandwich.

According to another aspect of the present invention,

There is provided a method for providing information for diagnosis of breast cancer or breast cancer metastasis, comprising the steps of:

(a) contacting an antigen specifically binding to an antibody immobilized on a support with a lectin to which an optical labeling substance recognizing an O-linked glycan of the antigen is contacted, thereby detecting the amount of lectin to which the optical labeling substance ; And

(b) measuring a signal of the optical label substance in which the lectin to which the optical label substance is attached is combined with the antigen.

The features and advantages of the present invention are summarized as follows.

(i) In the method of measuring cancer marker using the antibody and lectin of the present invention, various kinds of antigens can be measured regardless of the types of sugar chains exposed to the antigen.

(ii) The method of measuring the marker using the antibody / lectin of the present invention can be used as a substitute for the enzyme-linked immunoassay based on the measurement results corresponding to the conventional enzyme-linked immunosorbent assay.

Figure 1 is a schematic diagram of an antibody / lectin sandwich assay.
FIG. 2 is a specific schematic diagram of the antibody / lectin sandwich assay.
①: Support (gold electrode, silicon wafer)
②: Monolayer (DTSP, MUA, Cystamine)
③: capture antibody
④-1: Sugars included in the antigen (glucose, galactose, fucose, mannose, sialic acid, TF antigen)
④-2: Antibody recognition site (epitope)
⑤: Optical labeling materials (gold nanoparticles, colored polystyrene beads, quantum dots, colored dyes, fluorescent polystyrene beads)
⑥: Lectins (proteins that recognize and bind specific sugar chains, SNA, PNA, ConA, WGA)
Fig. 3 is a schematic view showing synthesis of lectin and fluorescent beads.
Fig. 4 is a graph showing the results of confirming the increase of fluorescent beads according to the concentration of CA15-3 (a): SNA lectin and (b): PNA lectin.

According to one aspect of the present invention,

(i) contacting an antigen that specifically binds to an antibody immobilized on a support with a lectin attached with an optical label substance recognizing an O-linked glycan of the antigen; And

(ii) measuring the amount of lectin to which the optical label substance in contact with the antigen is attached

Lectin < / RTI > sandwich for the measurement of breast cancer markers comprising the antibody-lectin sandwich.

Lectin is a protein that selectively binds to carbohydrates. It is isolated and purified from plants, microorganisms, viruses, invertebrates, vertebrates, etc., and is widely used as a material for various disease related research. About 300 species have been reported to date. Lectins are well known for their ability to aggregate and sediment red blood cells, classify blood types, and in recent years, they have various functions such as the ability to specifically coagulate cancer cells. That is, a lectin binds to a sugar having a specific structure, and makes selective exchange of information between a cell having a lectin substance and a cell having a sugar binding thereto.

In particular, in relation to cancer, the sugar chain on the cell surface has been found to be an important factor in determining the metastasis of cancer to the experimental model. This indicates that the lectins have different binding properties to the surface glycans of mutated cancer cell mutants, And the possibility of changing the metastasis by modifying the sugar chains of cancer cells has been confirmed from the results of studies such as the results of the studies. These properties are known to be common properties regardless of the type of cancer cells and organs that are targets of metastasis.

The immunoassay or immunohistochemical staining formats can be used in a variety of ways including, but not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, ≪ / RTI > and immunoaffinity purification. Methods of immunoassay or immunostaining include, but are not limited to, Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme - linked immunosorbent assay ( ELISA ), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e.g., ¹⁴ C, ¹²⁵ I, ³² P And < ^{RTI ID = 0.0} > S35) < / RTI >

The enzymes bound to the lectins include, but are not limited to, enzymes that catalyze a chromogenic reaction, a fluorescent reaction, a luminescent reaction, or an infrared reaction, and examples thereof include alkaline phosphatase,? -Galactosidase, Diphtheria oxidase, luciferase, and cytochrome P ₄₅₀ . When alkaline phosphatase is used as an enzyme binding to the lectin, it is possible to use bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS- B1-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- (10-acetyl-3,7-dihydroxyphenoxazone), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (N-methyl acridinium nitrate), resorpine benzyl ether, luminol, amplex red reagent tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronin, glucose oxidase and t-NBT (nitroblue tetrazolium) phenzaine methosulfate) may be used.

The detection lectin has a label for generating a detectable signal. The label may be a chemical (e.g., biotin), an enzyme (alkaline phosphatase,? -Galactosidase, horseradish peroxidase and cytochrome P ₄₅₀ ), a radioactive material (such as C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). A variety of labels and labeling methods are Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

The detection of such a signal indicates that the molecular marker is present in an unknown sample, which indicates that breast cancer has finally developed. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

The method of the present invention can be applied to flow cytometry (Ormerod, MG, ed. 1990, Flow Cytometry: A Practical Approach.IRL Press), magnetic-activated cell sorting (MACS) Stem Cells , 23: 477-482 (2005) or immunoaffinity purification methods (Ed Harlow and David Lane, Using Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999).

Antibodies that specifically bind to the molecular markers can be prepared by methods routinely practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Patent No. 4,816,567) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol . Biol, 222:. 58, can be prepared by 1-597 (1991)). The general process for manufacturing antibodies is Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference.

By analyzing the intensity of the final signal by the above-described immunoassay, it is possible to diagnose breast cancer.

According to a preferred embodiment of the present invention, the breast cancer marker is CA15-3, which is a breast cancer marker.

According to a preferred embodiment of the present invention, the specific sugar chains of the antigens are exposed to O-linked glycans and include glucose, galactose, fucose, mannose, sialic acid or TF antigen.

According to a preferred embodiment of the present invention, the optical labeling substance may be gold nanoparticles, colored polystyrene beads, quantum dots, colored dyes or fluorescent polystyrene beads.

According to a preferred embodiment of the present invention, the lectin is selected from the group consisting of Sambucus nigra agglutinin (SNA), Peanut agglutinin (PNA), ConA (Concanavalin A), LCH (Lens culinaris haemagglutinin), GNA (Galanthus nivalis agglutinin), RCA (Ricinus communis agglutinin ), AIL (Artocarpus integrifolia lectin), VVL (Vicia villosa lectin), MAL (Maackia amurensis leukoagglutinin), MAH (Maackia amurensis hemoagglutinin), UEA (Ulex europaeus agglutinin) or AAL (Aleuria aurantia lectin). More preferably, it is judged using SNA and PNA.

Table 1 lists the types of lectins that bind to specific sugar chains.

Mannos  Coupled Lectin
( Mannose binding lectins ) ConA Concanavaline LCH Lentil lectin GNA Snowdrop lectin Galactose, N- Acetylgalactosamine  Coupled Lectin
( Galactose  / N- acetylgalactosamine binding lectins ) RCA Ricin, Ricinus communis Agglutinin, RCA120 PNA Peanut agglutinin AIL Jacalin VVL Hairy vetch lectin N-acetylglucosamine-binding lectins
(N- acetylglucosamine binding lectins ) WGA Wheat Germ Agglutinin, WGA N- Acetyl neuraminic  Acid coupled lectin
(N- acetylneuraminic acid binding lectins ) SNA Elderberry lectin MAL Maackia amurensis leukoagglutinin MAH Maackia amurensis hemoagglutinin Fuchos  Coupled Lectin
( Fucose binding lectins ) UEA Ulex europaeus agglutinin AAL Aleuria aurantia lectin

According to a preferred embodiment of the present invention, the breast cancer marker is CA15-3, which is a breast cancer marker.

According to another aspect of the present invention,

There is provided a method of providing information for diagnosis of breast cancer or breast cancer metastasis comprising the steps of:

(a) contacting an antigen specifically binding to an antibody immobilized on a support with a lectin to which an optical labeling substance recognizing an O-linked glycan of the antigen is contacted, and measuring the amount of the lectin to which the optical labeling substance ; And

(b) measuring a signal of an optical label substance in which the lectin to which the optical label substance is attached is combined with an antigen; and when it is higher than that of a normal person, it is determined to be a breast cancer or breast cancer metastasis state.

CA15-3 is a marker that maintains a level of about 30 U / ml or less in the blood of a normal person and increases the blood concentration as the cancer progresses. Therefore, the number of beads judged by the assay can be compared with that of a normal person, and the value of CA15-3 in the blood can be converted to determine whether or not the patient is breast cancer patient.

FIG. 2 is a general schematic diagram of the system developed by the present research team, and is broadly divided into an electrode portion in which an antibody capable of specifically detecting a cancer marker is immobilized, and a measurement portion in which a cancer marker can be measured using lectin. In order to increase the specificity of the marker, an antibody capable of specifically recognizing and binding a marker to the surface of the electrode was immobilized. Antibodies were immobilized after forming a self-assembled monolayer using cystamine, DTSP, MUA or the like. Using a self-assembled monolayer, the antibody can be immobilized more uniformly and reproducibly. Thus, the antibody immobilized on the electrode recognizes the specific amino acid sequence (epitope) of the antigen and captures the cancer marker. The cancer marker captured by the antibody was exposed to the O-linked glycan at the site other than the epitope, and the lectin specifically recognizes and binds to the synthesized optically labeled substance to react with the exposed surface of the cancer marker. By measuring the optical signal generated at this time, quantitative measurement becomes possible.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

One. Example  1: Measurement of breast cancer measurement sensors and optical signals

Through the present invention, a sensor capable of measuring cancer antigen 15-3 (CA15-3), which is a breast cancer marker, was developed and the optical signal according to the concentration of CA15-3 was measured. CA15-3 is a marker that maintains a level of about 30 U / ml or less in the blood of a normal person and increases the blood concentration as the cancer progresses.

To confirm that the optical signal increases with the concentration of CA15-3, Biosens . Bioelectron (2011), 26, 3818-3824 synthesized lectin and fluorescent beads. The fluorescent beads were exposed to carboxyl groups on the surface, and synthesized by binding with an amine group of lectin using EDC / NHS reaction (Fig. 3). For the synthesis, 20 μl of 1 mg / ml EDC and 1.7 mg / ml NHS were added to the MES buffer solution containing 0.1% fluorescent beads for 15 minutes at room temperature. Thereafter, 100 쨉 l of 50 ml lectin was added and reacted at room temperature for 2 hours. After synthesis, 20 μl of a solution containing 2 mg / ml PEG and 1 M L-lysine was added to block the surface of the fluorescent beads that did not participate in the reaction, followed by reaction at room temperature for 2 hours. Thus, measurement using fluorescent beads synthesized with lectin is easy to count and quantify fluorescent beads immobilized on the chip surface.

2. Example  2: Diagnosis of breast cancer Assay

For the modification of the measurement surface for breast cancer diagnosis, the solution was refined using a solution containing sulfuric acid and hydrogen peroxide in a ratio of 4: 1 for 5 minutes. Then, the purified chip was reacted with 3,3'-dithiodipropionic acid di (N-hydroxysuccinimind ester) (DTSP) at 5 mM for 2 hours to form a monolayer, and then an antibody specifically recognizing CA15-3 Was immobilized at a concentration of 200 / ml at room temperature for 1 hour. To block the reaction of the remaining residues of DTSP, bicarbonate buffer (pH 9.5) containing 10 mM ethanolamine was reacted at room temperature for 10 minutes. Later, the sugar chain of the antibody was treated with sodium periodate to expose the aldehyde to the end of the sugar chain, and then bound to the L-lysine to prevent binding of the lectin to the sugar chain of the antibody. Then, for the measurement of CA15-3, the purified antigen was diluted to 0 to 50 U / ml with a PBS buffer, and a sample was prepared and reacted for 10 minutes on a chip immobilized with an antibody. After the lectin-modified fluorescent beads were reacted with the lectin-exposed chip, the optical signals corresponding to the concentration of CA15-3 were measured. As a result, it was confirmed that when there was no antigen, nonspecific reaction by lectin hardly occurred and fluorescent signal proportional to the concentration of antigen CA15-3 was measured (FIG. 4).

Claims (6)

(i) contacting an antigen that specifically binds to an antibody immobilized on a support with a lectin attached with an optical label substance recognizing an O-linked glycan of the antigen; And
(ii) measuring the amount of lectin to which the optical label substance in contact with the antigen is attached
Lectin < / RTI > sandwich for the measurement of breast cancer markers.
The method of claim 1, wherein the breast cancer marker is CA15-3, a breast cancer marker.
The method according to claim 1, wherein the specific sugar chain of the antigen is glucose, galactose, fucose, mannose, sialic acid or a TF antigen.
The method of claim 1, wherein the optically-labeled material is gold nanoparticles, colored polystyrene beads, quantum dots, colored dyes, or fluorescent polystyrene beads.

The method of claim 1, wherein the lectin is selected from the group consisting of Sambucus nigra agglutinin (SNA), Peanut agglutinin (PNA), ConA (Concanavalin A), LCH (Lens culinaris haemagglutinin), GNA (Galanthus nivalis agglutinin), RCA (Ricinus communis agglutinin) Artocarpus integrifolia lectin, VVL Vicia villosa lectin, MAL Maackia amurensis leukoagglutinin, Maackia amurensis hemoagglutinin, UEA (Ulex europaeus agglutinin) or AAL (Aleuria aurantia lectin).
A method for providing information for diagnosis of breast cancer or breast cancer metastasis comprising the steps of:
(a) contacting an antigen specifically binding to an antibody immobilized on a supporter with a lectin to which an optical label substance recognizing an O-linked glycan of the antigen is contacted to detect an amount of lectin to which an optical label substance binding to the antigen is attached ; And
(b) measuring a signal of the optical label substance in which the lectin to which the optical label substance is attached is combined with the antigen.

Images