CS262046B1 - Mouse lymphocytaric hybridome c-04 producing antibody against keratine 18 - Google Patents

Abstract

The present invention relates to mouse lymphocytic antibody-producing hybridoma keratin 13 series of animal species deposited in the Research Institute's hybridoma collection of Clinical and Experimental Oncology in Brno under the designation RICEO-C-04. Saaotna monoclonal RICEO-C-Ol Je hybridoma antibody suitable for use in eQzymoimunolo ^ ick radioimmunoassay of test tissues, cells and body fluids as well as for visualization keratin in cells at the optical level electron microscope and for immuno- isochemical detection of keratin 18 on tissue cuts.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of murine myeloma line P3-X63-Ag8> 653 and murine spleen lymphoid cells producing an antibody against human keratin 18.

To date, anti-keratin antibodies have been produced by repeatedly injecting keratin as an antigen into test animals. The serum of such immunized animals, taken after some time of antigen treatment, serves as a source of antibodies used mainly to identify keratin-containing cell structures. This procedure, called conventional immunization, has several drawbacks. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum varies and does not repeat in each individual organism. In addition to the antibodies to the desired antigen, the organism generally produces antibodies against the impurities of the antigen preparation, which must be removed from sera in a complicated manner, called saturation. In the case of antisera against one of the known keratins, which has been described in man 19, the situation is complicated by the considerable similarity of each keratin, so preparing a conventional antisera against a single keratin by saturation is extremely laborious and seldom leads to a satisfactory result. antisera are therefore very difficult to standardize and are based on production in a wide range of quality ·

For the manufacture of each batch, a substantially pure immunization antigen and other antigens are required to saturate the ballast antibodies against impurities or similar molecules such as vimentin, desmin or lamina.

Lymphocyte hybridomas producing anti-keratin antibodies have been prepared in several countries, and in some of them they are also commercially available and are beginning to be used in clinical practice.

Antibodies offered abroad, however, lack a unique combination of advantageous characteristics characteristic of the C-04 antibody, ie they do not combine narrow specificity for keratin 18 with a broad spectrum of reactivity with different animal species, as well as utility in various methods of administration including immunoblotting and immunT hiatochemistry on metacarne-fixed paraffin tissue sections / methods where many monoclonal antibodies offered abroad cannot be used at all / ·

The above mentioned drawbacks of the above-mentioned and used procedure are eliminated if a hybridoma producing a monoclonal antibody against keratin 18, deposited in the collection of hybridomas of the Research Institute of Clinical and Experimental Oncology in Brno, Yellow Hill 7, is available, under the designation RICE0-C-04.

Said hybridoma was obtained by a method known from the literature (Fazekas de St. Groth S.), Scheidigger D. Production of monoclonal antibodie: Strategies and tactics, J. Immunol. Meth. 35: 1, 21, 1980; Galfré G, Howe S, Milstein C, Butcher G.W, Howard U.C: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266:

550 -, 1977 / cloning a set of hybrid cells resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of BALB / c mice immunized with an isolated cytoskeletal preparation of human cells of epithelial origin ·

The advantage of C-04 lymphocyte hybridomas is that it produces a completely homogeneous antibody, i.e., a monoclonal antibody that is capable of specifically reacting with human keratin 18 as well as equivalent keratin of a number of species. · C-04 hybridoma can be cultured in vitro in media suitable for growing animal cells or in vivo conditions in the peritoneal cavity of BALB / c mice. hybridoma C-04 is suitable for long-term storage in cans stored in liquid nitrogen · Production of monoclonal antibody can be started after thawing of cell canned without the need of additional antigen · Monoclonal antibody produced by hybridoma C-04 is specific for keratin with relative molecular weight of approximately 45,000 referred to as keratin 18 and does not react with any other intermediate filament protein or other cytoskeletal proteins as determined by immunoblotting, enzyme immunoassay, indirect immunoperoxidase, and indirect immunofluoreaction methods. Pass was obtained after electro *

3,262,048 theoretic transmission of a wide range of cell and tissue lysates and isolated cytoskeleton preparations of various human and animal tissues and cell lines, after aeparation of protein components in a polyacrylamide gel in sodium dodecyl sulfate under reducing conditions. To determine the isoelectric point characteristic of the target keratin recognized by antibody C-04 /, the proteins of the above-mentioned lysates and cytoskeletal isolates were first subjected to isoelectric focusing, followed by the aforementioned polyacrylamide gel electrophoresis. After transfer of the split proteins to the nitrocellulose membrane, keratin 18 was specifically visualized (detected) using antibody C-04 followed by rabbit antisera against mouse horseradish peroxidase-conjugated immunoglobulins. The result was also confirmed by an enzymatic immunoassay, where C-04 was shown to bind to keratin 18 but not to vimentin, serum proteins or keratin 7, 8, 19 and others. Using the iraunoperoxidase reaction on tissue sections (confirmed by the results of the indirect immunofluorescence method - see below) of human and animal tissues, antibody C-04 was found to be reactive with epithelial tissues containing keratin 18 and non-reactivity with other tissues.

In indirect immunofluorescence using C-04 antibodies and subsequent incubation with rabbit or porcine antisera against mouse fluorescein-iso-thiocyanate-labeled immunoglobulins, only in vitro keratin-type filaments were bound in the cell types tested in vitro. Neither microtubules or microfilaments distinguishable from keratin after treatment with colcemid and cytochalazine B reacted with antibody C-04, also other structures of tested cells with the exception of keratin filaraents were negative.

Example 1:

To multiply RICE0-C-04 hybridoma cells in vitro, 1x10 6 hybridoma cells were introduced into a Legroux culture flask containing 20 ml DEEM culture medium with 10% fetal calf serum and antibiotics (see below). After four days of growth of the hybridoma cell culture, a culture medium supplemented with the raonoclonal antibody C-04 released into the medium by the hybridoma cells was recovered from the flask. The potency of the monoclonal antibody was tested by successive two-fold dilutions

- 4 2B2 04β culture medium containing monoclonal antibody in phosphate buffered saline (BBS) pH 7.4 and β 10% bovine serum · The antibody reacted with a specific antigen (isolated keratin 18) in a radloimmunological assay using a rabbit anti-mouse immunoglobulin antibody labeled isotope up to 1t512 dilution. The specificity of the obtained antibody was tested in a panel of tissue culture cells, namely indirect immunofluorescence using a porcine or rabbit antibody against mouse immunoglobulin labeled with fluorescein-iso-thiocyanate, and 1 following electrophoretic transfer of cell lysates and cytoskeletal preparations to a nitrocellulose membrane followed by detection indirect immunoperoxidase assay (see above). The results are summarized in Table 1.

1: Reactivity of C-04 antibody to cells of different lineages (+ strong positivity, - negativity, IP - immunofluorescence, IB - immunoblotting, V - viraentin, K - keratins marked with keratin catalog numbers, G - glial

acidic protein, D - desrain) · Species Tissue origin, Line name Medium type Reaction cell type filament IF IB human Rhabdomyo sarcoma RD D + V - AND and Gllom U 118 MG G + V - - Pibroblasts Primokultura IN - - T-lymphoblastoid lines MOLT-3 IN - -; Breast cancer MCF-7 ^K 8.18.19 + and + i Carcinoma urine. bladder T 24 * 4,8,18,19 + + Pharyngeal cancer Detroit 562 ^K 4,5,6,7,8, 17,18,19 + + Epidertnoid carcinoma A431 ^K 5,6,7,8,13, 14, 15,16,17,18, + + Cow Epithelium of the mammary gland BMGE-H ^K 7,8,18,19 + + Rat Mořisův hepatom MH ^K 8.18 + + Mouse Fibroblast 3T3 IN - - From the results shown in Table 1. Obviously, the antibody C-04 shows the keratin type intermediate (non-intermediate) filaments

different species while being negative on spiny filament types. The narrow specificity for keratin 18 has been clarified

5,282,048 in immunoblotting, where a strong reaction with keratin 18 was found, with the other keratin present being negative (see Example 2: tissue specificity).

Example 2:

The culture medium enriched for monoclonal antibody C-04 was obtained by culturing RICE0-C-04 hybridoma cells as in Example 1. The tissue specificity of the antibody reaction was tested by indirect iraunofluorescence assay and confirmed by indirect immunoperoxidase reaction, in both cases on tissue sections. in Table 2 were taken from surgical or autopsy material, tissue blocks of 0.5 x 0.5 x 1 cm were frozen in liquid nitrogen and stored at -70 ° C. · For testing, the frozen blocks were adapted (heated ) to -25 to -30 ° C and to a cryostat apparatus, cuts of 5-7 microns were prepared from each of them. Tissue sections were then transferred to histological slides, after drying in air, the sections were fixed for 10 minutes with a 1: 1 mixture of methanol and acetone precooled to -15 ° C and after washing the remaining fixative 2 x 10 min. in saline with phosphate buffer pH 7.4 (PBS), the monoclonal antibody C-04 was applied to the sections. After an incubation period of 1 hour, unbound antibody was removed from the section. The incubation (40-60 min.) was terminated by washing three times in PBS after 5 minutes, and the sections were allowed to stand at room temperature. until dry and the preparations are mounted eg in glycerin medium. The results of evaluating the response of antibody C-04 to selected tissues are shown in Table 2j between the results obtained by indirect immunofluorescence and indirect immunoperoxidase reaction, no differences were found, therefore these methods are not shown in Tab. No. 2 presented separately.

Table 2. Reactivity of C-04 antibody to normal tissues of different species (+ strong positivity, - negativity, V-vimentin, D-desrain, G-glial acid protein, N-neurofilaments, K-keratin with appropriate numerical index)

Table 2

Kind Tissue Human Tissue

Cartilage

Muscle

Brain

Cerebellum

Blood vessels

Liver - hepatocytes

- bile ducts Urine bladder - epithelium

Intestinal epithelium of the airway epithelium

Mezotel

Epithelium of the mammary gland

Gall bladder epithelium Eidermis (including epid. Planta

Epithelium of the tongue, esophagus

Cow Connective Muscle Vessels

Epidermis

Liver - hepatocytes

- bile ducts

Intestinal epithelium

Epithelium of the mammary gland

Bladder epithelium; Epithelium of the sweat glands Epithelium of the pancreas

282 048

Type filaraent Reaction

IN

In D

N + G

V + D ^K 8.18 ^{+ K} 7.8.18.19 ^{+ K} 4.5.7.8,13.18.19 ^{+ K} 7.8.18.19 ^{+ K} 8.18.19 ⁺ ^ 7 , 8,18,19 ⁺ ^ 7,8,18,19 ⁺ * 5,7,8,14,16,17,18,19 ^{+ K} 7,8,18,19 ⁺ pedis) K _{1t2>5f6> 9ř10 > 11f14t}

15.16 ^K 4,5,6,13,14,15,16,17,19 VD

D + V

K (but not K ₁₈ ) ^K 8.18 ^{+ K} 7.8.18.19 ^{+ K} 8.18.19 ⁺

K (including Κ _1θ ) +

K - - +

K - - ¹ +

K - ”- +

- 7 Table δ. 2 - continued

Species Tissue Medium filament type 262 046 Reaction hamster Vazivo IN Muscle D Blood vessels D + V Lymphoid tissue IN Epidermia TO (but not Κ ,, θ: > - Liver - hepatocytes TO including Κ ^ θ + - bile ducts TO _ n _ + Intestinal epithelium TO __ n _ + Rat Vazivo in Muscle D Blood vessels D + v Liver - hepatocytes TO (including Κ _1θ ) + - bile ducts TO _ M _ + Intestinal epithelium TO o. «_ + Epithelium of the mammary gland TO + Epithelium urine. bladder TO tt _ + Myě Vazivo in -, Muscle D - Blood vessels D + V - Liver - hepatocytes TO (including K ₁₈ ) + - bile ducts TO _ M __ + Epithelium of the mammary gland TO +

The spectrum of tissues (see Table 2) reacting with antibody C-04 shows that all tissues containing keratin 18 are positive, whereas all tissues lacking keratin 18 are negative, both in human tissues and in other species tested. Immunoblotting analysis after electrophoretic separation of protein lysates or cytoskeletal preparations confirmed that keratin 18 is the only keratin recognized by C-04 in epithelial tissues containing multiple keratin. Table 2 also shows the reactivity of C-04 with keratin 18 in various species, which is a relatively rare and advantageous property for monoclonal antibodies against keratin.

- 8 Example 3: 262 04β

In view of the possibility of practical use in differential diagnosis of tumors, the reactivity of C-C4 antibody on tissue sections prepared from different human tumors was tested by immunofluorescence and immunoperoxidase methods (see method description in Example 2).

Table 3 · Reactivity of antibody C-04 on tissue sections of human tumors (+ positive reaction, - negativity,

V - vimentin, D - desmin, G - glial acid protein, K - keratin with appropriate numerical index).

Tumor type Medium filament type Reaction

Sarcoma V (or V + D) - Melanomas IN - Lymphomas IN - Gliomy G - Carcinomas intestines ^K 8.18.19 + breast ^K 7,8,18,19 + lung R, 8.18.19 + stomach ^K 8.18.19 + ovaria ^K 7,8,18,19 + of the bladder ^ 4,7,8,13,17,18,19 + Hepatomy ^K 8.18 +

The results of determining the reactivity of antibody C-04 to various human tumors confirm the specificity of this antibody for tumors of epithelial origin that contain keratin 18. Together with negative results in other tumor types, this suggests the diagnostic cost of the test antibody at different tumor lesions.

RICEO-C-O4 hybridoma cells have a morphological pattern of typical myeloma cells. Under in vitro conditions they grow in the form of semi-suspension cultures. The basic culture medium is Dulbecco's Modification of Minimum Essential Medium (DMEM) supplemented with L-glutamine (3mM), penicillin, streptomycin and inactivated fatal calf Bér (10%) from Sokolnice u Brna. The hybridoma is cultured at 37 ° C, the mean generation time is 20.3 hours, and the modal chromosome count 6 months after hybridoma construction was 86. The antibody produced is a murine IgG1 class monoclonal immunoglobulin, specifically binding the keratin 18 epitope.

A monoclonal antibody produced by hybridoma RICE0 _C-f-04 reacts with the keratin variety of species and is useful in various assays, including immunohistochemistry on tissue sections, immunoblotting, enzymeimmunoassay and radioimmunoassay and immunofluorescence determination e.g. in cultured cells. Thus, the C-04 antibody is useful for the detection of keratin 18-containing cellular cytoskeletal structures in humans, but also in various farm and experimental animals. It will be used especially in routine differential diagnostics in the departments of pathological anatomy and oncology as an important tool to distinguish positively reacting adenocarcinomas from other types of tumors, such as sarcomas, melanoromas or lymphomas. It also offers the possibility to distinguish adenocarcinomas from carcinomas from multilayer squamous epithelium that does not contain keratin 18 (eg epidermis). Thanks to its properties, it can be used in diagnostics in the field of veterinary medicine as well as in a number of applications in research of animal cell biology, namely in the departments of health care, education, the Czechoslovak Academy of Sciences and the SAS. agriculture.

Claims (1)

RICEO-C-04 mouse lymphocyte hybridor producing a monoclonal antibody of the IgG1 class against keratin 18 of a number of animal species.