Method of human blood serum testing for oncomarker muc1

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immunotechniques. Method of human blood serum testing for OncoMarker MUC1 is offered for diagnostics of breast cancer (BC) by direct solid-phase bivalent immune photometric analysis using pair of homogeneous antibodies M3F1/M3B11. Formed antigen-antibody complexes are detected using Mab-peroxidase conjugate followed by introduction of stain TMB, and standard OncoMarker is human fat lactoglobule antigen MUC1. Method allows for primary diagnostics of BC stages II and III and for prediction of secondary process development (relapses), and for efficient therapy in determined diagnosis. Application of declared method allows to detect 56-63% BC patients BC by blood serum analysis.

EFFECT: possibility of primary diagnostics of BC stages II and III and prediction of secondary process development (relapses).

4 ex, 2 tbl, 3 dwg

 

The invention relates to biotechnology and clinical immunology, and is to develop a method for quantitative determination of the level of tumor marker (antigen) MUC1 in serum of human blood in the diagnosis of breast cancer (BC) by direct solid-phase dvuhsostavnogo immunofuorescence assay (ELISA).

Antigen MUC1 is a highly glycosylated transmembrane protein found in both normal and malignant epithelial cells (Biochim. Biophys. Acta, 1995, 1241: 407-424). The MUC1 molecule consists of three domains: cytoplasmic, transmembrane and extracellular. Extracellular associated with the membrane component of the antigen is characterized by high polymorphism. In normal physiological conditions polymorphic MUC1 antigen is expressed on the cell surface and performs various physiological functions (J.Nucl.Med.Allied Sci 1990; 34 (Suppl 3):151-162).

Part of membrane-bound antigen is a soluble form of the MUC1 antigen (sMUC1) and circulates in various body fluids. A soluble form of MUC1 was detected in breast milk, peripheral blood, human urine, and supernatant cultures of tumor cell lines (Biochim Biophys Acta. 1995; 1241:407-424; Int J Cancer. 1995; 63:412-418; Cancer Res. 1987; 47:5476-5482; Dis Markers. 1988; 6, 185-194).

According to the European society of oncologists antigen MUC1 Nar is do with removeemployee antigen (CEA) is one of the main tumor markers in the diagnosis of breast cancer (EGTM// Tumor markers in breast cancer - EGTM recomendation, 1999). The most studied member of the family MUC1 antigen CA 15.3.

It is known that the process of malignant growth is accompanied by increased levels of tumor markers in serum. In patients with adenocarcinoma detected increased levels of CA 15.3 serum, which correlates with the size of the tumor (Breast Cancer Res Treat. 1996; 37:209-216; Cancer Immun. Immunother. 1990; 31:269-273; J. Clin. Oncol. 1997; 15:2322-2328; Clin. Chem. 1997; 43:585-593). Antigen MUC1 plays a significant role in the metastatic process (J. Nucl. Med. Allied Sci 1990; 34 (Suppl 3): 151-162). However, the sensitivity of the primary diagnosis on the level of CA 15.3 serum of breast cancer patients is 15-35%, as low levels of tumor marker in the serum of the patient does not guarantee the absence of malignant process. At the same time, patients with progressive disease, relapse and metastasis, have persistently high levels of tumor markers (Br.J.Cancer, 63: 809-813), so detection methods SA recommended EGTM for research monitoring and predicting the course of breast cancer (Blood, 2000, v.96(9): 3147-3153).

The first commercial test kit for determining the level of MUC1 in serum was human system Centocor CA 15-3TMRIA (Malvern, PA) using two monoclonal antibodies specific for epitopes localized on the extracellular domain of MUC1 molecules (Hybridoma 1984; 3:223-226). The first monoclonal antibody (Mab) DF3 directed to PEP IGNOU part of the molecule (DTRPAGS); another Mab - 115D8 specific for the carbohydrate epitope of MUC1.

Based on the same Mab developed ELISA test system IMx CA 15.3 MEIA (Abbott) and Enzymun-Test CA 15.3 (Boehringer Mannheim), and methods for determining a tumor marker in the serum of human blood (J. Cancer, 2001, 7: 181-190).

As the closest analogue to consider the way Enzymun-Test CA 15.3 (J. Cancer, 2001, 7: 181-190), with a sensitivity of 13-14% in the diagnosis of breast cancer in the serum of the patient of the tumor marker CA 15.3. When implementing this method used the format of direct solid-phase dvuhsostavnogo ELISA and, optionally, avidin-bitenova system, based on the high affinity glycoprotein egg whites (avidin) to the molecule of Biotin. Application procedures biotinylate antibodies allows you to simultaneously make in wells treated with streptavidin, antigen MUC1, biotinylated Mab DF3 and conjugated with peroxidase Mab 115D8. Standard marker is an antigen CA 15.3, isolated from supernatants cell line ZR-75 (Ann. Clin.Biochem, 1999, 36: 579-586). Using biotinylated monoclonal antibodies has both advantages (high sensitivity ELISA)and disadvantages (additional work).

The task of the invention is to develop a more effective and sensitive method for quantitative determination of tumor-associated antigen MUC1 in which favorote human blood.

The problem is solved by developing the method of quantitative determination of tumor marker MUC1 in serum of human rights in the format of direct solid-phase dvuhsostavnogo immunofuorescence analysis using the test system of two monoclonal antibodies that interact with the tumor marker serum and in control with the standard marker; characterized in that the reaction of interaction of tumor marker-antibody is carried out in an ELISA format, as the test system uses a pair of monoclonal antibodies M3F1/M3B11 for the detection of the formed complexes antigen-antibody using Mab-peroxidase conjugate, followed by the introduction of dye TMB, and as a standard marker selected from the fat globules of the milk of human antigen MUC1.

Kit for determination MUC1 in serum of human blood.

The test system based on monoclonal antibodies M3F1/M3B11 is a standard set of components for direct solid-phase dvuhsostavnogo ELISA determination of MUC1 antigen (ELISA):

1) anti-MUC1 Mab 3F1;

2) standard antigen MUC1;

3) controls 1 and 2 with the contents of the antigen 8 and 16 ng/ml, respectively;

4) conjugate anti - MUC1 Mab MV with horseradish peroxidase (MW-RO);

5) buffer solutions for cultivation and application samples (phosphate buffer with a pH of 7.2, containing 0.05% bovine serum albumin and tween 20);

6) puff the RNA solution (phosphate-citrate buffer with pH 4.5) and the substrate for processing (3,3',5,5'-tetramethylbenzidine, TMB);

7) the solution to stop the reaction (10% solution of sulfuric acid).

The method in General.

The inventive method is based on direct solid-phase dvuhmetrovom ELISA. The test serum and the standard antigen added to wells pre-coated with Mab M3F1. Test serum diluted with buffer for drawing samples at a ratio of 1:40. Use no more than 25 μl of patient serum. The MUC1 antigen in the deposited serum samples and controls, specifically associated with Mab (M3F1) on the substrate, identified by applying Mab-peroxidase conjugate (MU-RO). Next to the development of the color reaction in the wells add substrate mixture containing as a colorant TMB, and measure colour intensity spectrophotometrically at a wavelength of 450 nm. The amount of antigen in the tested serum samples determined by a calibration curve constructed for a standard antigen MUC1. A standard curve is built for each analysis in coordinates: optical density versus concentration of antigen in the standard (Figure 1), where: X is the concentration of antigen MUC1 (1-64 ng/ml); Y-axis is the optical density at 450 nm. When determining in the sample more than 16 ng of antigen serum sample grow further in 2-4 times.

Interpretation of test results is done using the estimated average level of MUC1 antigen in healthy is onerow (cutoff) The ELISA Guidebook, pp.301-394 in "Methods in Molecular Biology" (v.149), Humana Press, 2000). Serum containing the MUC1 antigen at a concentration above the cutoff level, diagnosed as pathological. The range of normal values MUC1 antigen set based on local environmental factors and objectives of the study. In the present method apply two-level cutoff, depending on the purpose of the study: cutoff 1 - monitoring of treatment and to detect recurrence, a cutoff of 2 for the primary diagnosis of breast cancer (see Example 4).

The main parameters for the quantitative determination of MUC1 by the claimed method.

The main parameters for the quantitative determination of MUC1 correspond to GOST R 51352-99 and presented in table 1. Cross-reaction with bilirubin, hemoglobin, human, and when using sera of patients with lipemia as possible sources of interference is not detected (Method Validation-The Interference and Recovery Experiments, 2000).

Table 1
The parameters for GOST R 51352-99The requirements of GOSTThe results of determination by the claimed method
The reproducibility (coefficient of variation)No more than 15%5-10%
The limit of detection of MUC1 antigen2±0.1 ng/ml
Linearity90110% 95-110%
% open90-110%95-115%

Immunospecificity feature a panel of monoclonal antibodies to MUC1.

Mab M3F1 and MW receive as a result of hybridization of normal lymphocytes immunized mouse (Balb/c) myeloma cells (P3/X63-Ag8) and characterized by the ability to connect with the natural mucin isolated from milk (MUC1), recombinant polypeptide VNTR, Hypo - and deglycosylation the mucin - deMUC1 (residual carbohydrate content <1%; the content of sialic acid - 1%), and native mucin tumor cell line carcinoma person Kzt47 D.

Strains - producers of monoclonal antibodies PMBC N-97 (M3F1) and PMBC N-98 (MV) deposited in Russian national collection of industrial microorganisms. (Patent of the Russian Federation 2006130857 and 2006130858, respectively).

The invention is illustrated in the following figures. Figure 1. A standard curve determine the number of antigen MUC1 by the claimed method.

Figure 2. The distribution of marker levels (MUC1, ng/ml) among donors and patients in the determination of the claimed method.

Figure 3. ROC curves for the proposed method (examined 71 patients with a primary diagnosis of breast cancer II-III stages) and Enzymun-Test CA 15.3 (examined 77 patients with II-III stages of breast cancer).

Example 1. Getting conjugate Mab MSW with horseradish peroxidase.

2-4 mg peroxidase XP is on (Amressco, conjugation grad, USA) is subjected to oxidation periodates sodium and unite with detalizovannye Mab MW in the ratio of 1:2 (J.Histochem.Cytochem, 1974, 22, 1084). The reaction is stopped by tetraborate sodium (4 mg/ml at 4°). Received peroxidase conjugate cialiswhat against phosphate buffer pH 7.4 and subjected to salting out with a saturated solution of ammonium sulfate. Suspension is centrifuged 20 min at 5000g and dissolve the precipitate in borate buffer (pH 8.0). The conjugate is stored at 0°after adding glycerol in a 1:1 ratio. Working dilution obtained peroxidase conjugate is 1:2500.

Example 2. Obtaining standard antigen MUC1.

MUC1 is obtained from the fat globules human milk method immunoaffinity chromatography on brazian sepharose 4 (Sigma, USA)conjugated with specific to MUC1 Mab (MS), followed by alyazia 0.1 M glycine (modification of the method Burshell J.et al./Cancer Invest., 1989, 17, 53-61). The resulting antigen MUC1 is stored in lyophilized form with a concentration of 5 mg/ml at 4° or in the form of ready-to-use solution with the addition of preservative (0,2% thimerosal).

1 ng of the obtained antigen MUC1 corresponds to 2 U SA antigen 15.3 (where U is the universal unit according to the international classification MUC1 antigen).

Example 3. The definition of MUC1 levels in the sera of blood donors and patients with breast cancer stages II and III. The calculation of the cutoff.

Studied 111 the sample further machining work : the sera. Of these, 41 serum of healthy women, 52 - breast cancer patients stage II, 18 - stage III breast cancer patients. The sample size of normal and pathological sera determined by formulas based on the given values of specificity and sensitivity of the method, The ELISA Guidebook, pp.301-394 in "Methods in Molecular Biology" (v. 149), Humana Press, 2000).

The content of MUC1 in serum of healthy women in the determination of the claimed method ranges from 3.5 to 16 ng/ml, and in some cases reaches and 20 ng/ml Average statistical content antigen MUC1 in normal is 10 ng/ml with a standard deviation of 3.5 (p=0,95).

To select the cutoff value used graphical method, The ELISA Guidebook, pp.301-394 in "Methods in Molecular Biology" (v. 149), Humana Press, 2000; Radiology, 2003, 228: 3-9).

The results determine the level of MUC1 in serum of breast cancer patients and healthy donors present in the form of a diagram (Figure 2), where: X represents the interval content of antigen in serum from 5 to 40 ng, and the Y - axis the number of samples serum, the content of the MUC1 antigen which corresponds to this interval. The cutoff value is determined by conducting vertical lines through the point of intersection of two arrays: the area of concentration of normal and pathological sera. Thus, the cutoff in the present method of determining the marker lies in the range of 10-15 ng/ml

Cutoff 1 is equal to 15 ng/ml, which coincides with the value of this parameter is near what about the analogue of (30 U MUC1/ml, 1 ng=2 U). The level of antigen MUC1 below cutoff1 have 93% of tested donors. 56% of breast cancer patients in phase II and III have the level of tumor marker above cutoff1.

To reduce the proportion of false negative results, which is important when the primary diagnosis of cancer, use a cutoff of 2, equal to 12 ng/ml (figure 2). 63% of breast cancer patients in phase II and III have the level of tumor marker above cutoff 2 and the proportion of false-negative diagnosis of breast cancer is reduced by 7%.

Example 4. Calculation of the parameters of the diagnostic efficiency of the proposed method, depending on cutoff 1 and 2.

Based on the results of the quantitative determination of MUC1 tumor marker in the serum of healthy and diseased breast cancer carry out the calculation of sensitivity, specificity and other parameters the effectiveness of the proposed method (table 2) The ELISA Guidebook, pp.301-394 in "Methods in Molecular Biology" (v. 149), Humana Press, 2000).

50% of the sera of patients II and 67% of stage III breast cancer have a level marker above cutoff 1. Diagnostic sensitivity for stage II breast cancer increase to 61% by using a cutoff of 2.

In the diagnosis of breast cancer stages II and III independently selected cutoff effectiveness of the proposed method is several times higher than the nearest equivalent.

Comparing the effectiveness of the proposed method of diagnosis and the closest analogue (Enzymun-Test CA 15.3) when breast cancer is also often done by constructing ROC curves (Radiology, 2003, 228: 3-9), and the walking of that is, the larger the area under the curve, the higher diagnostic efficiency of the method. X-axis figure 3 - value of (1 - specificity) at a cutoff of 5 to 30 ng/ml, Y - axis sensitivity of ELISA test system for specified cutoff frequency and specificity. For comparison used ROC curve closest analogue according to published data (Clincal.Chemistry, 1997, 43: 4, 585-593). The inventive method of determining marker MUC1 is more efficient than the nearest equivalent as at a specificity of at least 70% has a higher than the nearest equivalent sensitivity (75% and 40%, respectively) (Fig 3)

Table 2.
ELISA systemCutoff (a)Se, % (b)Sp, % (C)And, % (d)In, % (e)E % (f)
M3F1/M3B1115 ng/ml5693916172
12 ng/ml6383836472
Enzymun-Test CA 15.330 U/mlthe 13.493,98035,840,4
(a) the level of MUC1 antigen in healthy donors;

(b) the sensitivity of diagnostics;

(c) the special which was mentioned diagnostics; < / br>
(d) the proportion of patients diagnosed with a similar diagnosis to detect the specified method;

(e) the proportion of healthy, with a negative result in the determination by the specified method;

(f) efficiency ratio diagnostic method.

For the proposed method note:

- higher accuracy positive () and negative (B) test results (83-91% and 61-64%, instead of 80% and 36% at the nearest analogue, respectively);

- 2 times higher efficiency ratio diagnostics (E), that is the correct classification of patients and healthy;

increasing the specificity of the method to 93% and a reduction of false-negative results when predicting recurrence and metastasis in the case of cutoff 1;

to increase the sensitivity of the method to 63% in the case of cutoff 2 with a primary diagnosis of stages II and III breast cancer.

From the presented data it follows that the inventive method of determination of MUC1 tumor marker in the serum of human blood has a high diagnostic efficiency, allowing for its use in clinical practice to identify 56-63% of patients in the primary diagnosis of breast cancer stages II and III, monitoring disease recurrence and assess the effectiveness of therapy when the diagnosis.

The method of quantitative determination is of marker MUC1 in serum of human rights in the format of direct solid-phase dvuhsostavnogo immunofuorescence analysis using the test system of two monoclonal antibodies, interacting with the tumor marker serum and in control with the standard marker, characterized in that the test system uses a pair of monoclonal antibodies M3F1/M3B11 for the detection of the formed complexes antigen-antibody using Mab-peroxidase conjugate, followed by the introduction of dye TMB, and as a standard marker selected from the fat globules of the milk of human MUC1 antigen.



 

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