Antigenic peptides, a method for detecting secreted by tumors particles

 

(57) Abstract:

Describes new antigenic Petey from secreted by tumors particles (LP), having the amino acid sequence provided in the sequence of the protein 100 kDa of these TLP with one of the following sequences: (a) Arg-Thr-Asn-Lys-Glu-Ala-Ser-Ite; b) Asn-Gln-Arg-Asn-Arg-Asp. Describes the method of detecting secreted by tumors of the particles. 3 S. and 3 C.p. f-crystals, 2 Il.

The invention relates to the fields of peptides protein complexes of human TLP (proteins secreted from tumors) with antigenic activity, and antibodies reactive with these proteins that will be used in diagnostic and clinical purposes.

Complexes TLP are protein complexes that are present in human tumor cells, especially in cells of carcinoma of the lung; among proteins TLP described protein of 100 kDa (Tarro C., "Oncology" 40, 248-253, 1983). TLP isolated from tumor tissues, as described in EP 283433, the description of which is incorporated herein by reference. Very useful to have diagnostic tests (methods of analysis) to identify such complexes or their fractions of crude extracts of lungs.

The author's attempts to obtain specific antibodies against whole is him, to get specific reagents.

The author of this invention identified peptide sequence protein TLP 100 kDa, having antigenic activity, received specific antibodies and demonstrated that these antibodies specifically react with the TLP from carcinoma of the lung.

Accordingly, an object of this invention is an antigenic peptide secreted by tumor particles (TLP), having the amino acid sequence contained in the sequence of protein 100 kDa from TLP.

In the preferred implementation of the invention the specified peptide has at least one of the amino acid sequence listed at the end of the description, as Th ID N 1, Last ID No. 2 or the Last ID No. 3. Preferably, these peptides are synthetic or natural source.

In further carrying out the invention the specified peptide contains a cysteine residue or carboxyl, or amine end group of the molecule and a carrier covalently associated with the specified residue; preferably specified molecule, the carrier is hemocyanin, most preferably specified hemocyanin obtained from oysters.

Granvania antigenic peptide secreted by tumor particles (TLP), having the amino acid sequence contained in the sequence of the protein TLP 100 kDa.

Preferably these antibodies recognize a peptide having at least one of the amino acid sequence listed at the end of the description, as Th ID N 1, Last ID No. 2 or the Last ID No. 3.

An additional object of this invention are diagnostic kits for determining the TLP from samples containing antibodies of this invention, as specific reagents.

Another object of this invention is a method of determining complexes TLP in the samples, which includes stages, as follow:

- thus the specified pattern using the first amount of serum to the TLP;

the detection of such complexes TLP in the specified immunoprecipitating the material through their reaction with the second specified number of serum to TLP and by means detects the specified reaction.

This invention will be described hereinafter with reference to some illustrative, but not limiting examples of which include accompanying drawings, wherein:

Fig. 1 represents some graphics RIA immunological assays serum samples from 4 orlitat direct Western blot turns using serum 419 (1) and 428 (2), as well as pre-immunized sera (4-6) on samples of lung extracts;

Fig. 2B shows the result of Western blot turns when the extracts were pre-immunoprecipitation using sera 419 and 428; and

Fig. 2C shows the result of Western blot turns, when the extracts were pre-immunoprecipitated as described in example 3.

Example 1. The discovery and synthesis of peptides from protein TLP.

Complex TLP isolated from extracts of carcinoma of the lung, as described in EP 283443. Protein is blocked on the terminal amino group and, accordingly, is cleaved by V8 protease from Staphylococcus aureus using known techniques. One of the resulting peptide fragments represents the following sequence:

XaaXaaArgThrAsnLysGluAlaSerIle

Two synthetic peptide was synthesized using solid-phase method using a peptide synthesizer 430 from Applied Biosystems as described in M. Bodanszky, Springer Verlag, New York, N. Y, 1984, with the following sequence:

5'-ArgThrAsnLysGluAlaSerIleCys-3'

5'-CysArgThrAsnLysGluAlaSerIle-3'

The identity and purity of the product were confirmed by analysis of amino acids and HPLC (using the technique of liquid chromatography high pressure).

Using, ederland, 1988, these peptides are associated with cysteine sulfhydryl end groups with a side lysine residue of hemocyanin oysters when using maleimidomethyl-N-hydrocycloning ester (MBS) as a reagent.

Example 2. The immunization.

Four rabbits were injected subcutaneously with 0.5 g of a mixture of complexes from example 1 in 1.5 ml of the FSB (phosphate saline buffer) and 0.5 ml of complete adjuvant's adjuvant. Incomplete adjuvant as activating the injection was administered through a two-week intervals. Serum samples were collected every two weeks and subjected to RIA, which is described in "Laboratory Techiques in Biochemistry and Molecular Biology", 4th edition, by T. Chard, Elsevier Science Publishers, Amsterdam, The Netherlands, 1990, using 96-microtiter cell boards, covered with known concentrations of synthetic peptides as antigens. Board incubated with dilutions of serum at different levels, then washed and treated iodirovannoi protein A to detect the presence of specific antibodies in serum. Fig. 1 represents the data obtained with serum samples from four immunized rabbits as described above. All sera show a peak of radioactivity approximately 15,000 counts/min at a dilution of 1:1000 is yserowanej sera as controls, in the range of 1000 pulses/min up to 1500 pulses/min, showing a ratio of 10:1 at a dilution of 1:1000. Relation to the background signal above 20:1 for serum 419 and 428.

Example 3. Reaction with sera.

Serum 419 and 428 were analyzed using extracts of the lungs, which are obtained as described in EP 283443, from three patients suffering from pulmonary carcinoma epidemiologi type or adenocarcinoma, according to the "International Classification of Diseases for Oncology", 2nd edition, 1990, edited by C. Percy, V. Van Holte and C. Muir, WHO, Geneva (Fig. 2A). Samples of these extracts (1 and 4 = B. C.; 2 and 5 = S. G., 3, and 6 = M. R.) diluted solution of detergent and divide by the method of polyacrylamide gel electrophoresis in accordance with known techniques. Then there was a direct Western blot turns using serum 419 (1) and 428 (2). Found two nonspecific protein bands of approximately 55 kDa and 35 kDa, which are also present when using pre-sensitized serum (4-6).

Then the extracts immunological precipitious sera 419 and 428 with the use of appropriate methods, which are described by E. Harlow and D. Lane, Gold Spring Harbor Laboratory, Gold Spring Harbor, N. Y. (1988); immunological precipitiously material is separated on the gel and uses the OSA 100 kDa is detected in all the extracts in the analysis using serum 419 (1-3), and serum 428 (4-6). The analyzed extract 1 and 4 demonstrates the presence of a very large number of protein TLP.

To determine whether the reaction is specific, performed the following control tests, which are presented in Fig. 2C, where: line 1 represents the serum of the patient SU, and immune serum 419 is used or thus or for Western blot turns; line 2 is obtained by using a pre-sensitized serum for stage and thus serum 419 for Western blot turns; line 4 is the result of the use of serum 419, pre-incubated with the peptide with the Last ID No. 1 to stage precipitation and serum 419 for Western blot turns; line 5 is obtained using serum 419 for stage and thus pre-sensitized serum for Western blot turns.

The results confirm that a protein band of approximately 100 kDa specifically reacted with immune serum 419.

Sequence listing

(2) Information for sequence ID No. 1:

(I) sequence Characteristics:

(A) Length: 8 amino acids

(B) Type: amino acid

(D) Topology prirodnye source:

(A) Organism: Homo sapiens

(F) tissue Type: carcinoma of the lung

(XI) sequence Description: Pet ID # 1:

< / BR>
(2) Information for sequence ID No. 2:

(I) sequence Characteristics:

(A) Length: 7 amino acids

(B) Type: amino acid

(D) Topology: linear

(II) molecule Type: peptide

(III) Hypothetical: no

(VI) a Natural source: Homo sapiens

(A) Organism:

(F) tissue Type: carcinoma of the lung

(XI) sequence Description: Pet ID N 2:

< / BR>
(2) Information for sequence ID No. 3:

(I) sequence Characteristics:

(A) Length: 6 amino acids

(B) Type: amino acid

(D) Topology: linear

(II) molecule Type: peptide

(III) Hypothetical: no

(VI) a Natural source of:

(A) Organism: Homo sapiens

(F) tissue Type: carcinoma of the lung

(XI) sequence Description: Pet ID # 3:

< / BR>
Example 4. Obtaining a peptide of the neoplastic tissue.

Neoplastic tissue after removal of necrotic material and after washing in a solution of phosphate buffer crushed and homogenized with repeated cooling on ice, the homogenate after three freeze and thaw-rayed at the preliminary centrifugation at 3000g scheme, listed at the end of the description, and the precipitate (1) preliminary centrifugation after homogenization, ultrasonic treatment and subsequent centrifugation at 3000g forms a supernatant (2) and sediment (2), and sediment (2) subjected to the same procedure, forms a precipitate (3), which is thrown away, and the supernatant (3), which is added to supernatant (1) and (2);

b) centrifuging the total supernatant collected from supernatant (1), (2) and (3) at 100000g and concentration of the supernatant;

C) location (after the test on the activity) of the supernatant to the column with isoelectric focusing and collecting fractions of the peak, check their activity, and

d) an increase in the degree of purification using chromatographic column.

Toxicity studies.

The results of the research products Timely A (TLPAand Timely B (TLPB) Roman firm IFI, allow to draw the following conclusions:

A) Products Timely A (TLPAand Timely B (TLPB) in normal laboratory animals:

1) have very low levels of mortality and acute toxicity in rats and dogs, in connection with the various methods of introduction into the body (subcutaneous, intraperitoneal, intravenous) are well tolerated even increase ctice;

2) repeated introduction into the body of dogs and rats does not lead to significant changes the most important hepatic and biochemical constants (azotemia, glycemia, peripheral blood, hematopoietic brain tissue, system urine formation, aspartate aminotransferase, serum, glutamine-pyruvic transaminase level of serum alkaline phosphatase serum) as well as the histological structure of the main bodies;

3) at doses in kg, exceeding the dose recommended in clinical practice, do not provoke the dog, rabbit and rat significant changes in blood pressure, respiration and ECG;

4) does no harm to females, waiting for posterity, not have a negative impact on pregnancy and embryo development and fetal rats and rabbits at doses per kg of weight, sometimes exceeding the possible dose used in clinical practice (0.5 - 1 mg in adults).

In connection with the foregoing we can conclude that, with respect to the usual laboratory animals products Timely A (TLPAand Timely B (TLPBare drugs, which even at doses exceeding the dose used in clinical practice, do not show sharp on the ASS="ptx2">

Synthesis of antigens.

On the basis of the previous data sequence of the protein were prepared by two synthetic peptide using methods of solid-phase peptide synthesis (method of Merrifield) on the same group Applied Biosistems 430. Synthesis of the peptides BOC-amino acids. The hydrolyzed peptides from the resin, without the protective properties using liquid HF and purified liquid chromatography high resolution. The identity and purity of the product is confirmed by mass spectrometry plasma desorption, amino acid analysis and analytical HPLC. Cysteine residues are included to attach to the protein-carrier. Since it was not known in advance which of them will give the best serum titers were derived peptides with cysteine on both ends. They were individually attached to hemocyanin fissurella using aminobenzoyl maleic acid - N - oxenfree acid. This reagent provides the accession of aminoacetic on the side chain of lysine in the media to the sulfhydryl of the cysteine on the peptide.

Extraction of Timalia of tumor cells.

1. Rinse 1 confluent T-300 1 x DPBS and dry.

2. Add approximately 2 ml of lytic residence time of surface growth, centrifuged for 15 min at 15000 rpm.

Lyse buffer: 50 mM Tris-Cl pH of 7.4, 50 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% of Triton X-100, 0.1 mM Navo3, 0,4 mM Pefablos.

3. Remove supernatant and install the concentration of protein.

4. To stratify and freeze fraction for multiple distillation gel to prevent decomposition.

SDS - page/transfer.

1. Enter 60 mg of Timalia protein lysate in the gel with 10 - 20% of Tricine (NOVEX) and to disperse at a voltage of 125 V, 80 mA to the unit gel until the dye front reaches the gel precipitate.

2. Transfer protein in SC at 175 mA for about 2 hours.

3. Block for one hour with 5% milk in 1X TBS - tween 20, gently shaking at room temperature.

4. Dilute 1oAB 1:200 in 1% milk, 1X TBS-T (i.e., 100 ml IHH anti-Timelive - 1 mg/ml in 20 ml 1% milk, 1 X TBS-T). To stand all night at t - 4oC, shaking.

5. Rinse 3X in 1 X TBS-T for 10 min each wash.

6. Dilute 2oAt 1:2000 in 1% milk, 1 X TBS-T (i.e., 10 units of peroxidase from horseradish antireligioso IHH, connected to a solid At - Amersham # NA-934 in 20 ml of 1% milk, 1 X TBS-T). To stand for 2 hours at room temperature, shaking.


1) sizes between 48,1 and 61 (based on mol. weight),

2) have a lipid group (based on elution with concavalin A),

3) isoelectric point less than 7.0 (based on the characteristics of elution on Sephadex DEAEA with 0.2 M NaCl).

Tumors obtained from the surgical Department of the Monaldi hospital, Naples, Italy, was investigated as follows.

Fresh tumor tissue with an approximate weight of between 25 and 150 g were separated from the necrotic areas and were carefully rinsed 20 mm Tris-HCl buffer, pH to 7.2. This procedure was performed at 4oC under sterile conditions to reduce the possibility of infection, prior to subcutaneous administration in vivo and in vitro experiments performed tests for sterility and progenote. After this treatment, the fabric was finally rasmalai scissors, punching through a stainless steel sieve with holes the size of 60 mesh. and resuspendable in 20 mm Tris-HCl buffer, pH 7,2 ( in ratio 1:2).

The suspension is then centrifuged at 1500 rpm for obtaining intact cells.

Sediment cells, resuspending, as described above, then three times quickly saturazione of 0.9 kHz for 9 min on ice. The material treated by ultrasound, which was defined as the concentration of the protein was centrifuged at 100000 g for 1 h at 4oC.

After ultracentrifugation the precipitate was discarded and the supernatant samples of 0.5, 1.0, 1.5 and 2.0 ml were taken to determine immunological activity.

The Sephadex G-100.

The material obtained in the previous phase were subjected to gel filtration on Sephadex G-100. Briefly, 60 mg of the supernatant was layered on the gel, pre-balanced 20 mm Tris-HCl buffer (pH 7,2) and suirable the same buffer, with the speed of elution of 8.0 ml/h of the Collected fractions of 2 ml, the optical density was measured at a wavelength of 280 nm and peak fractions were tested on the subject of immunological activity.

Isoelectric focusing.

In accordance with the present invention is proposed extraction TLP proteins in the procedure which removed the deficiencies at all stages of improved methods of purification, which includes isoelectric focusing (IEF) in addition to the earlier separation technique, consisting in high-speed centrifugation, in which consistently collect supernatant to obtain okonchatelnom chromatography on a column of DEAE And (Sepharose). The physical properties of the extracted material is preferably determined on the columns of gel IEF and SDS page with DDS/Sephadex in the case of the determination of molecular weight and glycosidic groups. Antigenic proteins (TLP), obtained in accordance with the present invention, in essence, represent lipoglycopeptide with the following distinctive features:

a) dimensions between 48,1 and 61 angstroms (defined on the basis of known data about their molecular mass);

b) the presence of a lipid group (shown on the basis of the characteristics of elution from concanavalin A);

C) solubility and

d) isoelectric point less than 7,0 (defined on the basis of the characteristics elution with DEAE And Sephadex (1 anion) (elution with 0.2 M NaCl).

Electrophoresis with polyacrylamide gel with sodium dodecyl sulfate (SDS page with VAT).

Electrophoresis was performed in SDS page with 0,1% VAT. Briefly, after denaturation of the protein by adding DDS 2-mercaptoethanol and bromophenol blue (as a marker dye) to a final concentration of 0.1, 0.1 and 0.5% respectively, the connection boiled for 2 minutes Samples with glycerol at a final concentration of 10% was layered on tubes with 6% SDS page, and electrophoresis was carried out at t the lar mass after staining of the gel and washing was determined by linear regression analysis of log10molecular weight marker proteins from mobility relative to bromophenol blue.

Ion-exchange chromatography.

Immunoreactive material after gel filtration was chromatographically on DEAE-Sephadex a-50, pre-balanced 20 mm Tris-HCl (pH of 7.2), using the column size 1.5 x 30 cm and the initial volume of the gel 20 ml 5 ml sample with a protein concentration of 15 mg/ml was applied on the column, then suirable 20 mm Tris-HCl buffer (pH 7,2) as the first eluting buffer. Then applied the step elution using 0.4 and 0.8 M NaCl in the same buffer, the noise at a rate of 30 ml/h Usually switch to a second buffer, the authors present invention was carried out only after the absorption of light at 280 nm prior eluate returned to the control level. Fractions of the eluate 2 ml were collected and the peak fractions were pooled and tested them both on the concentration of protein and immunological activity.

Determination of antigen TLP in the serum of a person using enzyme-linked immunosorbent assay (ELISA). Diagnostic kit.

Serum samples of 50 μl were incubated with 100 μl biotinylated CSH-419 IgG (rabbit) immobilized on Board the Finance of the new complex on the principle onestepatatime sandwich method: TLP immobilized IgG/TLP/TLP labeled IgG. After the formation of the complex was added chromatogr and the substrate. The intensity of the generated signal was proportional to the amount of TLP contained in the sample (measurement was performed using an ELISA reader ETI System at 450 nm). Tablets for micrometrology streptavidin coated, buffer, Chromogen and substrate were provided by the company Sorin Biomedica to create a test set that is suitable for industrial production. Purified nonapeptide CHS-275 associated with BSA (CSH-275/BSA), can be determined by this set, showing linear dependence on serial dilutions of non-specific binding (HCO) controls (data not shown). As agents for calibration was used pools of sera (their reactivity was determined by curve CHS-275/BSA); agent for calibration with the lowest determined by the reactivity was chosen to establish the resolution of the method used in each definition. Agent for calibration with OP, similar to the OP BSA (0,07 - 0,11), was used as negative control. The resolution of the method must be higher than the mean + ICO (standard deviation) of the negative control.

Serum values of OP > values resolution of sposobna the following tests: a) test the linearity of the relationship between serum with known high content of TLP and values of OD; b) assessment of the reproducibility of the method by combining two studies in each experience 5 different sera; C) assessment of the reproducibility of the method by exploring 2 different serums in 10 different assays. All serum samples were examined blindly.

The statistics. Chi-square test was used to compare the differences between the two groups. Differences were considered significant when the P value < 0,05.

1. Antigenic peptides from secreted by tumors particles (TLP), having the amino acid sequence provided in the sequence of the protein 100 kDa of these TLP with one of the following sequences: (a) Arg-Thr-Asn-Lys-Glu-Ala-Ser-Ile; b) Asn-Gln-Arg-Asn-Arg-Asp.

22. Antigenic peptides under item 1, with synthetic origin.

3. Diagnostic kit for the detection of redundant tumor particles (TLP) in the sample containing the first specific reagent for TLP-antibody capable of selectively recognize and bind antigenic peptide under item 1, the second reagent are able to recognize and associate the specified antibody; a detection tool.

4. Diagnostic kit according to p. 3, characterized in that the second reagent is an anti-antibody (IgG).

6. The way to detect secreted by tumor particles TLP of the samples, including the contacting of the extract of the tumor tissue with the antibody having specific activity for the investigated antigen, characterized in that the receiving immune serum with antibodies to antigenic peptides under item 1, are immunoprecipitate extract tissue carcinoma of the lung with these sera, allocate complexes TLP in immunoprecipitation material by reaction with a second specified number of serum to TLP using immunobloting.

 

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