The method of obtaining specific antisera to universal tumor antigen and a method for the diagnosis of malignant tumors using this antisera

 

(57) Abstract:

The invention relates to medicine, namely to Oncology, and relates to a method of obtaining specific antisera to universal tumor antigen and method for the diagnosis of malignant tumors using this antisera. The invention includes the preparation of cell suspensions from the embryo at the stage of fetus genetically homogeneous animals, repeated immunization of animals of the same genetic line of this suspension, the cells of the spleen, the selection of lymphocytes, immunization of animals with the same genetic line suspension of these lymphocytes and obtaining antisera, which is added to the body fluids of a subject, and the results, significantly different from control, diagnose the tumor. The invention improves the sensitivity and versatility of the method of diagnosis. 2 S. and 2 C. p. F.-ly.

The invention relates to medicine, more precisely to the cancer, its sections and to the diagnosis of malignant tumors.

A brief overview of the immunoassay in Oncology shows the following.

In 1949, L. A. Zilber first showed, and in 1957, So Pran and j.Maine confirmed that the cells of slocate the P CLASS="ptx2">

1) viral Antigens of tumors. They are identical for any viral tumors of this type.

2) Antigens carcinogenic tumors. They are strictly individual for patients and tumors.

3) Isoantigen transplant type or TSTA - tumor-specific transplantation antigens different in all individual tumors induced by chemical agents and identical in different tumors caused by a single virus.

4) Embryonic antigens.

In the process of carcinogenesis cells are dedifferentiate, acquiring embryonic type of structure. They often find embryonic antigen-specific embryonic stages of development of the organism. These antigens are able to immunize the body against tumors. The most studied antigens: - fetoprotein and removeability antigen (CEA). The I-th detect when primary carcinoma of the liver, II - with adenocarcinoma of the colon, stomach, esophagus or pancreas.

In children with neuroblastoma, lymphosarcoma or brain tumors detected2- fetoprotein, cancer of the stomach - fetal Californication. These antigens are localized in cell membranes or circularise. They cannot be attributed to alien for the body, because in addition to the tumor they are present in other normal tissues. Among them is renal antigen, which is present in normal kidney and in liver tumors - hepatoma.

Adenocarcinoma of the kidney contains the antigen lungs and liver.

Immunodiagnostics of malignant tumors based on the indication in the blood of patients with the above antigens, antibodies and detection sensitized to tumor antigens to lymphocytes.

On detection of a-fetoprotein based metaly diagnosis of lymphosarcoma, neuroblastoma (see On the detection of antibodies to CEA - how to patent RF N 2077725, CL G 01 N 33/53, leukosis virus - the method according to the author's certificate N 1641443, G 01 N 33/53).

On the detection of heterogeneous antigens RF patent N 2063768, 1991, a 61 K 39/00, RF patent N 2025734, G 01 N 33/53, ed. mon. USSR N 1589215, G 01 N 33/53, author's certificate N 1704087, G 01 N 33/53, ed. mon. N 170922, ed. mon. N 1589215.

In AVT. mon. N 1805392 (G 01 N 33/53) described a method for diagnosing cancer by antigens (HLA-B35) lymphocytes.

However, the existing level of diagnostics is that, in fact, none of the tests is not universal. Detection in blood antic the left and tissue antibodies. There are no methods to identify specific universal tumor antigen.

In this regard, prospective identification of sensitized lymphocytes that inhibit the growth of colonies of tumor cells. However, they are active only against "his" type of tumor.

Thus, the methods used immunodiagnostic tumors weakly satisfy the requirements of the primary tumour diagnosis, is totally unsatisfactory screening of malignant tumors and high-risk groups and can be applied with certain limitations, only immunomonitoring treatment of malignant tumors.

The failure of the existing methods can be explained by the fact that the used antisera to tumor antigens, strictly speaking, are not.

The present invention is to obtain antisera to the universal tumor antigen irrespective of the type of tumor and organ.

The prototype as claimed method of producing specific antisera, and method of diagnosis using the selected method according to the patent of Russian Federation N 2063768, 1991, a 61 K 39/00, which includes the selection of the tumor tissue from deceased persons, Zamora the supernatant, immunization of animals with the extract, the blood of immunized animals, obtaining from it the product, the introduction of specific antisera reactive with the blood of the subject, the results of which diagnose the tumor.

The claimed method differs from known allows you to get anticigarette to idiotope T-cell receptor functioning in cells of malignant tumors, i.e. antiidiotypic antiimperialism serum that provides diagnostics of all types of tumors, regardless of their origin and location.

For the method of obtaining the specific antisera, you must conduct a two-step immunization, highlight the embryo at the stage of Fetus genetically homogeneous animals, dispersing it, to prepare a cell suspension. Cell suspension to provide immunization of an animal of the same genetic line. Then you need the immunized animal to extract cells of the spleen, to separate the lymphocytes in the density gradient of ficoll-urografin (1,065-1,079). These lymphocytes should conduct multiple immunization of syngeneic intact animals and get them in a standard way anticigarette. This anticigarette follows prefilterattribute with different types of tumors, received from different people and in different organs.

It allowed, on the basis of the obtained antisera to develop a method of diagnosis of malignant tumors. Known similar methods for diagnosing tumors have a high enough sensitivity and even the most effective of them, it shall not exceed 40-60%. This low sensitivity of known cancer immunodiagnostics tests due to the fact that in these reactions the tumor markers, strictly speaking, are not and are organ-specific or oncofetal antigens, inherent in the normal individual organisms or organs. This leads to the fact that the expected universal immunological expression features a common mechanism of tumor formation is replaced private inherent in non-neoplastic conditions (inflammation, collagen).

It is known that organ-specific antigens are not required for neoplastic transformation of cells, and that gives such a high percentage of false-positive results in the diagnosis of malignant tumors.

The proposed method of detection of the marker is fundamentally different from currently used that determine the x tumour progression.

The method is based on the results of theoretical and experimental work of the author, in which it is established, that in any histologically different tumour cells functioning sustainable in the tumor progression process T-cell immunological detection surface embryopathies antigens and that this mechanism underlies the phenomena of tumor formation (immortality and progression).

For the implementation of the method for the diagnosis of tumors is necessary to prepare a specific anticigarette proposed method it is received, enter anticigarette to universal tumor antigen in immunological reaction with the tissues or body fluids of the subject, and then the reaction immunofluorescence assay or reaction ESR to diagnose the tumor. As tissues can be used tumor tissues in response immunofluorescence assay or blood of the patient in response ESR.

Diagnosis of the tumor is determined during the statistically significant differences of the results of the reactions between the test and control samples.

In the case of the reaction of the ESR for calculating the difference between the test and control samples ispolzuyetsa 1,5;

A - the value of ESR in the experimental sample (citrate to blood added anticavity to the antigen of the tumor);

B1and B2- the value of ESR in the control samples (citrate blood added to the serum of the same species of animal that was used to obtain antisera);

X - the maximum value of ESR in the analysis (or in the sample A or medium B1and B2, i.e., (B1+ B2)/2.

An example implementation of the methods of obtaining antisera and diagnosis of malignant tumors.

The Wistar rats weighing 300-500 g extracted embryo at the stage of the Fetus. Cloth of his dispersed in medium 199 in the ratio of the volume of fabric:medium 199 1: 5. The obtained suspension was carried out weekly immunization of intact Wistar rats. 1.5 months have scored rats extracted spleen, dispersed and gradient ficoll-urografin 1,065-1,079 derived lymphocytes.

One of them was prepared for suspension cells:medium 199 in the ratio of 1:1, which was administered weekly other intact rats. After 5 immunizations rats clogged, they have taken blood, "clarified" derived from her anticavity, filtered, with the specified anticorodal supplied reaction ESR in the following groups bmcl 5% buffered solution of sodium citrate added 800 μl of fresh venous whole blood, taken at the time of analysis, no later than 20 seconds after the fence. From the time of mixing the blood with a preservative prior to analysis should not be more than 1 h In the presence of hemolysis or clotting analysis can not be set. From this blood take 3 servings 70 ál each in 3 separate tubes. In one type working (with antibodies) in 2 other control (without antibodies) in the serum of 20 ál each. Serum injected directly into the blood with a preservative, and not on the walls. Capillaries must be of the same size. The mixture is stirred and fill their capillaries to the level of 5/0. Stand 1 hour After 1 hour remove the indicators and cheated them by the above mathematical formula.

In this way was obtained in Wistar rats anticavity used in the diagnosis of disease in individual patients.

In the analysis of blood of the patient-OIC, 1942 birth, d-s: colorectal cancer following results:

A = 25, B1= 28, B2= 28

On a mathematical formula found coefficient

< / BR>
1,7 > 1,5, i.e., the diagnosis of malignancy is confirmed.

In the blood of a patient with fibroids earlobe obtained the following results:

A = 10,a malignant tumor is confirmed.

Below are the results of the research in the group of patients with malignant tumors.

BREAST CANCER is the 125 patients, sensitivity - 83,2%

LUNG CANCER - 247 patients, the sensitivity of 98.1 per cent

STOMACH CANCER - 156 patients, the sensitivity of 85.2%

CANCER of the COLON - 23 patients, the sensitivity of 82.5%

RECTAL CANCER 27 patients, the sensitivity of 92.5%

THYROID CANCER - 58 patients, the sensitivity of 79.5%

KIDNEY CANCER - 38 patients, the sensitivity of 78.6%

CANCER of UTERINE BODY - 412 patients, the sensitivity of 75.0%

CERVICAL CANCER - 41 patient, the sensitivity of 81.8% of

CONTROL GROUP -

Healthy - 400 people, sensitivity to 5.1%

Fibrocystic breast disease - 221 people, sensitivity to 8.3%

Gastritis - 120 people, sensitivity to 6.2%

Peptic ulcer - 62, sensitivity to 8.3%

Collagen - 40 people, sensitivity to 6.5%

Pneumonia (acute. and khron.) 60 people, sensitivity to 7.2%

Prostatitis - 18 people, sensitivity is 2.1%

Chronic colitis - 115, the sensitivity of 4.2%

Conclusion: the proposed method has a sensitivity of not less than 85,9% and specificity not manageworkflow tumor antigen in the application is the main component of the diagnostic drug, sold in Russia and abroad under the brand name TURTESTR.

1. The method of obtaining specific antisera to universal tumor antigen, including the selection of fabrics, preparation of cell suspensions, immunization of animals, the blood of immunized animals, obtaining from it the target product, characterized in that the quality of the fabrics at the first stage of genetically homogeneous animals use the embryo at the stage of the fetus, prepare cell suspension, conduct repeated immunization of this suspension, produce the cells of the spleen, perhaps, lymphocytes, conduct subsequent immunization of animals of the same genetic line suspension these lymphocytes are anticigarette and filter it.

2. The method according to p. 1, characterized in that the filtering is made through porous filters.

3. A method for the diagnosis of malignant tumors using specific antisera to universal tumor antigen, wherein the add anticigarette under item 1 or 2 to the tissues, blood or other body fluids of subjects with subsequent consideration of the results of immunofluorescence, in reactions ESR or other known methods immunodefi the manual on p. 3, characterized in that the reaction results erythrocyte sedimentation rate calculated by the formula

,

where is the diagnostic factor, which in the presence of the tumor is 1.5;

A - the value of ESR in the experimental sample (citrate to blood added anticavity to the antigen of the tumor);

B1and B2- the value of ESR in the control samples (citrate blood added to the serum of the same species of animal that was used to obtain antisera);

X - the maximum value of ESR in the analysis (or in the sample A or medium B1and B2, ie ).

 

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FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

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