KR100309141B1 - Immunoassay using 5'-deoxy-5'-methylthioadenosine - Google Patents

Abstract

PURPOSE: Provided is an immunoassay for diagnosing cancer according to the detected amount of 5'-deoxy-5'-methylthioadenosine, thereby diagnosing large intestine cancer, stomach cancer, breast cancer and rectal cancer easily. CONSTITUTION: The immunoassay for diagnosing cancer is composed of preparation of an antibody for 5'-deoxy-5'-methylthioadenosine and detection of the quantity of 5'-deoxy-5'-methylthioadenosine in blood or serum of a cancer patient by using enzymatic immunoassay such as radio immunoassay and enzyme-linked immunosorbent assay.

Description

Immunoassay using 5'-deoxy-5'-methylthio adenosine

The present invention relates to an immunoassay method for diagnosing cancer tumors according to the amount of 5'-deoxy-5'-methylthio adenosine (hereinafter referred to as MTA). More specifically, the present invention is a method of MTA through immunological analysis such as radioimmunoassay (hereinafter referred to as RIA) or Enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) for samples taken from biological tissues such as rectum, stomach, etc. The present invention relates to an immunoassay method that can be used for diagnosing cancer tumors by revealing the detection concentration.

5'-deoxy-5'-methylthio adenosine (MTA) is a naturally occurring substance in the nucleus during polyamine synthesis in all animal cells. Unlike polyamines, MTA does not accumulate in cells because MTA is rapidly degraded to methylthioribose-1-phosphate and adenine by MTAase, which is abundantly present in all normal cells and cell-lines differentiated from normal tissues. . The MTAP gene encoding MTAase is located on the p21-22 bane short arm of chromosome 9 and contains a type I IFN gene cluster, tumor suppressor genes CDKN2 (p16), CDKN2B (p15) and CpG islands. MTAase is reduced because the tumor suppressor genes, CDKN2 (p16), CDKN2B (p15), remove genes from each other. However, p16-deficiency cancer acts on malignant tumor cells, human leukemia cells, and tumor cells to delete MTAP gene. For example, 40% in melanoma cells, 57% in giant cells of lung cancer, 71% in glioma and 78% in lucemia cells. On the other hand, Kaneco et al. (1984) found that more MTA is detected than normal cells by HPLC analysis of samples taken from urine from leukemia and melanoma. Therefore, the measurement of MTA shows the possibility of clinical use as a cancer tumor marker.

Accordingly, an object of the present invention was devised in view of the above facts, and established a systematic method of antibody preparation and immunoassay in a small amount of biological sample, and a small amount of serum, etc., using enzyme-linked immunosorbent assay such as RIA or ELISA. The purpose of the present invention is to provide an immunoassay method for diagnosing malignant tumors, cancers, leukemia cells, etc. by measuring MTA concentrations in biological samples.

The object of the present invention is to set up a systematic method of antibody preparation and immunoassay method, and reacting a small amount of samples extracted from living cells with antibodies, and then comparing and analyzing the detected amounts of MTA using an immunoassay such as RIA or ELISA. Is achieved by doing so. Hereinafter, the configuration and operation of the present invention.

1 shows absorption spectra of MTA-BSA conjugates.

Figure 2 is a photograph showing the 10% SDS-PAGE results of the MTA-BSA conjugate.

3 shows the detection amount of MTA according to the RIA measurement.

Figure 4 shows the affinity comparison of anti-MTA antiserum with ribose and various nucleic acids.

Figure 5 shows the concentration of MTA according to the RIA serum collected from normal people and cancer patients.

Figure 6 measures the concentration of MTA from the cell medium of the cells obtained from Leukemia cells.

7 shows MTA measurement results according to ELISA.

The present invention comprises the steps of preparing an antiserum and immunogen for MTA; Comparing and analyzing the detected amount of the MTA prepared by the antiserum and immunogen prepared by the Competitive RIA and ELISA method.

Bovine serum albumin (BSA), 5'-deoxy-5'-methylthio adenosine (MTA), DTT, sodium periodate, sodium borohydride, ethylene glycol, Freund's complete adjuvant, Freund's used in the following examples and experimental examples incomplete adjuvant, 1-ethyl-3- (dimethylaminopropyl) -carbodiimide (EDC) and goat anti-rabbit IgG were purchased from Sigma, USA, and S-adenosyl-L- [methyl- ³ H Methionine was purchased from Amalsham, UK, and the microtiter plate was obtained from Costar, avidin-peroxidase, biotin hydrazide, 2,2'-azine-di [3-ethyl Bezthiazoline-sulfonate] was purchased from Pierce, RPMI 1640 media, heat-inert petalbobin serum, L-glutamine, penicillin, streptomycin, and the like were purchased from GIBCOBRL, USA. Hereinafter, the configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Preparation of Immunogen and Antisera

A conjugate of protein and MTA was prepared according to Erlanter and Beiser's method (1964). That is, 15 mg of MTA was added to 50 mg of sodium periodate dissolved in 2 mg of water, and stirred and mixed in the dark for 60 minutes. Excess periodate was removed by adding 5 µl of pure ethylene glycol. After 5 minutes to the mixture was added BSA (bovine serum albumin) solution, KLH (keyhole impet haemocyanine), poly-L-lysine (2.5mg / ㎖ DW) was added to K ₂ CO ₃ was adjusted to pH 9.0 ~ 9.5 range. The mixture was chromatographed on a Sephadex G-50 column equilibrated with 1 mM DTT solution and phosphate buffer saline (PBS) to remove free MTA to obtain protein-MTA conjugate as an immunogen. In this case, whether the protein-MTA was conjugated and the free MTA was removed through absorbance analysis as shown in FIG. 1. That is, the concentration of BSA is determined according to the Bicichoninic acid (BCA) protein crystallization method, and free MTA has an absorbance of 260 nm (FIG. 1c), so that the junction of MTA-BSA is determined to have an absorbance of 240-300 nm. In addition, 10% SDS-PAGE analysis of the MTA-BSA conjugate showed a molecular weight ranging from 30 kDa to 200 kDa. Meanwhile, BSA (bovine serum albumin) solution, KLH (keyhole impet haemocyanine), and poly-L-lysine (2.5mg / ml DW) were added to each of three rabbits in turn, and after 4 weeks, they were added twice, and finally, After 10 days, rabbit blood was collected. The collected blood was centrifuged to obtain antiserum, and a conjugate of MTA and BSA was added to rabbits to prepare an antiserum.

Example 2. RIA Measurement

S-adenosyl-L- [methyl- ³ H] methionine (91Ci / mmol) was acid hydrolyzed and heated for 10 minutes to prepare [methyl- ³ H] MTA. Distilled water was added to this reaction solution at a ratio of 1: 200 to dilute the reaction solution, and each diluted aliquot was stored at -20 ° C. Samples isolated from various biological tissues such as rectum and stomach were mixed with 1.2 M perchloric acid at a volume ratio of 1: 3 and the pH was adjusted to 7.0. In this mixture, those with molecular weights of 10,000 or more are filtered out with Centricon. The antiserum prepared in Example 1 was diluted 10-fold and 20 μl of the diluted antiserum was added to the mixed reaction solution prepared above. Then, 100 μl of standard solution or 50 μl of sample was added to a total of 190 μl of the mixed reaction solution. Heated at 37 ° C. for 30 minutes with 10 μl of MTA (91Ci / mmol) labeled with ³ H]. After 30 minutes, 200 μl of 100% saturated ammonium sulfate solution was added to each sample and left at 4 ° C. for 20 minutes. Each sample tube was centrifuged at 13,000 × g for 15 minutes, the supernatant was separated, washed with 50% saturated ammonium-sulfate and centrifuged. As a result of radiographic measurement of the centrifuged precipitate, as shown in Figure 3, the lowest detection amount was 100 pmol per tube, the range of operation was 0.5 ~ 100 pmol per tube. In addition, the results of the experiment in Example 1, the addition of DTT in the production of the antibody was found to be more effective to use BSA instead of KLH.

Example 3. Reproducibility Experiment of MTA by RIA

MTA concentrations were analyzed by RIA in five stages ranging from 3.0 to 11.3% using livers of liver, leaf axis of soybean, and union of soybean. As a result, as shown in Table 1 below, the concentration of MTA in the cells was in the range of 0.5-0.7 nmol / g.

Result of reproducibility analysis of MTA by RIA Sample nmol MTA ± SD / tissue 1g % CV ^a n ^b rat liver 0.606 ± 0.049 8.1 5 soybean axes (1 day) 13.543 ± 0.667 4.9 5 soybean axes (2 day) 6.798 ± 0.482 7.1 5 soybean axes (4 day) 1.012 ± 0.030 3.0 5 soybean callus 0.275 ± 0.031 11.3 5

(In Table 1, CV ^a represents the coefficient of variation, n ^b represents the number of determination)

Example 4. Antibody Specificity Assay

Specificity of anti-MTA-BSA antiserum was measured by measuring the affinity of adenosine, AMP, ADP, ATP, adenine, adenosyl homocysteine, SIBA, etc., having a structure similar to that of [methyl- ³ H] MTA bound to the antibody. As a result, as shown in Figure 4, the anti-MTA-BSA antiserum did not cross-react with adenosine, AMP, ADP, ATP, adenine, and ribose, but cross-reacted with SIBA.

Example 5 Detection of MTA in Serum and Culture Medium

Experimental Example 1. Cell Culture

K562, CCRF-CEM and HeLa cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. At this time, 2 mM L-glutamine, 100 IU / ml penicillin and streptomycin were added and performed in a 37 ° C. 5% CO ₂ incubator.

Experimental Example 2. Measurement of free MTA levels in serum of cancer patients and normal subjects

Samples obtained from serum of normal and cancer patients and cells obtained from the cell culture of Experimental Example 1 were examined for free MTA levels by RIA. As a result, as shown in FIG. 5, the MTA level of the sample collected from normal and cancer patients was 1-30 pmol / ml, and above all, the MTA level was detected in the samples of colorectal cancer, gastric cancer and rectal cancer patients. In addition, MTA up to 100 pmol / ml was detected in culture medium of CRE-CEM and K562 Cell, a kind of leuchemia cells obtained from cell culture.

Example 6. Biological MTA-BSA Conjugate Preparation

100 μl of biotin hydrazide 50 mM is added to 4 ml of BSA-MTA conjugate and mixed with 60 μl of EDC. This reaction involves an EDC-mediated conjugate between the biotin hydrazide and the carboxylic acid of MTA-BSA. Carbodiimide reacts with carboxyl groups to form O-acrylurea intermediate activity. After overnight incubation in the greenhouse, centrifuge at 13,000 g for 30 minutes and remove the precipitate. The supernatant is placed in a Sephadex G-50 column and protein fragments are collected.

Example 7 Indirect Antigen Binding ELISA Assay

Indirect antigen binding ELISA measurement was performed using the biotin-bound MTA-BSA and avidin-peroxidase prepared in Example 6. In other words, the immunoplates are coated with rabbit anti IgG in 35 mM bicarbonate buffer and incubated overnight at 4 ° C. After the coating process the plates are washed three times with PBS and partitioned with 1% BSA-PBS for 1 hour in the greenhouse. The plates are washed with PBS to purify test samples or standard aliquots with anti-MTA IgG and biological MTA-BSA conjugates are added to each well. The well plate is incubated at 4 ° C. overnight and washed three times with PBST. 100 μl of peroxidase substrate (ABTS) solution is added to each well. The absorbance change was measured by a microtiter plate spectrophorometer using a 405 nm filter. As a result, as shown in FIG. 7, the minimum limit detection amount of MTA was 100 pmole.

As described in detail in the above embodiments, the cancer tumor diagnosis method according to the detection amount of 5'-deoxy-5'-methylthio adenosine (MTA) of the present invention can easily diagnose colorectal cancer, gastric cancer, breast cancer and rectal cancer. It is a very useful invention in the biopharmaceutical industry.

Claims (3)

In the cancer tumor analysis method, an antibody against 5'-deoxy-5'-methylthio adenosine (MTA) is prepared, and then an enzyme immunoassay is used to measure the concentration of MTA in the blood or serum of cancer patients. Cancer tumor immunoassay method. The cancer tumor immunoassay method of claim 1, wherein the enzyme immunoassay is selected from one of a radioimmunoassay and an enzyme-linked immunosorbent assay. The cancer tumor immunoassay method of claim 1, wherein the cancer tumor is selected from the group consisting of colorectal cancer, gastric cancer, breast cancer and rectal cancer.