CS262046B1 - Mouse lymphocyte hybrid 3-04 producing anti-keratin 18 antibody - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma, producing an antibody against keratin 13 of a number of animal species, stored in the hybridoma collection of the Research Institute of Clinical and Experimental Oncology in Brno under the designation RICEO-C-04. The monoclonal antibody of the hybridoma RICEO-C-01 is suitable for use in enzyme-immunological and radioimmunological analysis of tested tissues, cells and body fluids, as well as for visualization of keratin in cells at the optical and electron microscope level and for immunohistochemical detection of keratin 18 on tissue sections.

Description

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

Up to now, antibodies against keratin have been produced by repeatedly injecting keratin as an antigen into experimental animals. The serum of animals immunized in this way, collected after a certain period of exposure to the antigen, serves as a source of antibodies, used mainly for the identification of cellular structures containing keratins. This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable in each individual organism. The organism usually produces, in addition to antibodies against the desired antigen, antibodies against impurities of the antigen preparation, which must be removed from the serum in a complex manner called saturation. In the case of antisera against a single one of the group of known keratins that have been described in humans so far 19/ the situation is further complicated by the considerable mutual similarity of the individual keratins, so that preparing a conventional antiserum against a single keratin by saturation is extremely laborious and rarely leads to a satisfactory result. Production batches of conventional antisera can therefore only be standardized with great difficulty and come out of production with a wide range of quality.

For the production of each batch, it is necessary to prepare a highly pure immunization antigen and other antigens to saturate ballast antibodies against impurities or against similar molecules such as vimentin, desmin or lamins.

Lymphocytic hybridomas producing antibodies against keratins have already been prepared in several countries, and in some of them they are also commercially available and are starting to be used in clinical practice.

However, antibodies offered abroad lack the unique combination of advantageous properties characteristic of the C-04 antibody, i.e. they do not combine narrow specificity for keratin 18 with a broad spectrum of reactivity with various animal species, as well as usability in various application methods including immunoblotting and immunohistochemistry on metacarn-fixed paraffin tissue sections (i.e. methods in which many monoclonal antibodies offered abroad cannot be used at all).

The shortcomings of the above-mentioned and previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against keratin 18 is available, stored in the hybridoma collection of the Research Institute of Clinical and Experimental Oncology in Brno, Žlutý kopec 7, under the designation RICE0-C-04.

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

550 — , 1977 / by 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 the lymphocytic hybridoma C-04 is that it produces a completely homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with human keratin 18, as well as with equivalent keratins from a number of animal species. The hybridoma C-04 can be cultivated in vitro in media suitable for growing animal cells or in vivo in the peritoneal cavity of BALB/c mice. Hybridoma C-04 is suitable for long-term storage in cans stored in liquid nitrogen. Monoclonal antibody production can be initiated after thawing the cell preserve without the need for additional antigen. The monoclonal antibody produced by hybridoma C-04 is specific exclusively for keratin with a relative molecular mass 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 immunofluorescence. The proof was obtained after electro*

- 3 262 048 ioretic transfer of a wide spectrum of cell and tissue lysates and preparations of Isolated cytoskeleton of various human and animal tissues and cell lines, after separation of protein components in polyacrylamide gel in sodium dodecyl sulfate environment under reducing conditions. To determine the isoelectric point >characteristic for the target keratin recognized by the C-04 antibody/, the proteins of the above-mentioned lysates and cytoskeletal isolates were first subjected to isoelectric focusing, followed by the already mentioned polyacrylamide gel electrophoresis. After transfer of the separated proteins to a nitrocellulose membrane, keratin 18 was specifically visualized (detected) using the C-04 antibody and subsequently rabbit antiserum against mouse immunoglobulins conjugated with horseradish peroxidase. The result was also confirmed by an enzyme immunoassay, where the binding of the C-04 antibody to keratin 18 was demonstrated, but not to vimentin, serum proteins or keratins 7, 8, 19 and others. Using the irradionoperoxidase reaction on tissue sections (also confirmed by the results of the indirect immunofluorescence method - see below) of human and animal tissues, the C-04 antibody was found to be reactive with epithelial tissues containing keratin 18 and non-reactive with other tissues.

In indirect immunofluorescence using C-04 antibodies and subsequent incubation with rabbit or pig antiserum against mouse immunoglobulins labeled with fluorescein-isothiocyanate, binding occurred only to keratin-type intermediate filaments in the tested cell types in vitro. Neither microtubules nor microfilaments distinguishable from keratins after treatment with colcemid and cytochalasin B reacted with C-04 antibody, and other structures of the tested cells with the exception of keratin filaments were negative.

Example 1:

In order to propagate RICE0-C-04 hybridoma cells in vitro, 1x10^ hybridoma cells were inoculated into a Legroux culture flask containing 20 ml of DEEM culture medium with 10% fetal calf serum and antibiotics (see below). After four days of hybridoma cell culture growth, the culture medium enriched with the C-04 monoclonal antibody released into the medium by the hybridoma cells was obtained from the flask. The efficacy of the monoclonal antibody was tested by serial two-fold dilution

- 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 the specific antigen (isolated keratin 18) in a radioimmunoassay using isotope-labeled rabbit anti-mouse immunoglobulin antibody up to a dilution of 1:512. The specificity of the obtained antibody was tested on a panel of cells from tissue cultures, i.e. in indirect immunofluorescence using swine or rabbit anti-mouse immunoglobulin antibody labeled with fluorescein-isothiocyanate, and after electrophoretic transfer of cell lysates and cytoskeletal preparations to a nitrocellulose membrane with subsequent detection by indirect immunoperoxidase assay (see above). The results are summarized in Table 1.

zabulka no. 1. Reactivity of antibody C-04 with cells of different lineages (+ strong positivity, - negativity, IP - immunofluorescence, IB - immunoblotting, V - viraentin, K - keratins marked with numerical indices of the keratin catalog, G - glial

acid protein, D - desrain)· Type Tissue origin, Line name Type of medium Reaction cell type filament IF IB person Rhabdomyosarcoma RD D + V - I i Gllom At 118 MG G + V - - Pibroblasts Primoculture In - - T-lymphoblastoid lineage MOLT-3 In - - ; Breast cancer MCF-7 ^To 8,18,19 + i + i Bladder cancer T 24 *4,8,18,19 + + Pharyngeal carcinoma Detroit 562 ^To 4,5,6,7,8, 17,18,19 + + Squamous cell carcinoma A431 ^To 5,6,7,8,13, 14, 15,16,17,18, + + Cow Mammary gland epithelium BMGE-H ^To 7,8,18,19 + + Rat Mauris hepatoma MH ^To 8.18 + + Mouse Fibroblast 3T3 In - - From the results shown in Table 1. it is clear that the antibody C-04 shows keratin type intermediate filaments

of various animal species, while it is negative for osteitic filament types. Narrow specificity for keratin 18 has been specified

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

Example 2:

The culture medium enriched with monoclonal antibody C-04 was obtained by culturing hybridoma cells RICE0-C-04 as in Example 1. The tissue specificity of the antibody reaction was tested by indirect immunofluorescence test and also confirmed by indirect immunoperoxidase reaction, in both cases on tissue sections. The tissue samples listed in Table 2 were taken from surgical or autopsy material, tissue blocks measuring 0.5 x 0.5 x 1 cm were frozen in liquid nitrogen and stored at -70°C. For the actual testing, the frozen blocks were adapted (warmed) to a temperature of -25 to -30°C and sections of 5-7 micrometers thick were prepared from each of them on a cryostat. The tissue sections were then transferred to histological glass slides, after air drying the sections were fixed for 10 minutes with a mixture of methanol and acetone (1:1) pre-cooled to -15°C and after washing out the remaining fixative for 2 x 10 min. in phosphate-buffered saline pH 7.4 (PBS), the monoclonal antibody C-04 was applied to the sections. After an incubation period of 1 hour, the unbound antibody was removed from the section by repeated rinsing (3 x 5 minutes each) in PBS and rabbit or pig antibody with fluorescein isothiocyanate was applied to the section. Incubation (40 - 60 min.) was completed by washing three times in PBS for 5 minutes each, the sections were left at room temperature until dry and the preparations were mounted, e.g. in a medium with glycerin. The results of the evaluation of the reaction of the C-04 antibody with selected tissues are presented in Table 2j. No differences were found between the results obtained by indirect immunofluorescence and indirect immunoperoxidase reaction, therefore these methods are not presented separately in Table 2.

Table No. 2. Reactivity of antibody C-04 with normal tissues of different species (+ strong positivity, - negativity, V- vimentin, D - desrain, G - glial acidic protein, N - neurofilaments, K - keratin with the corresponding numerical index)

Table No. 2

Type Tissue human Connective tissue

Cartilage

Muscle

Brain

Cerebellum

Blood vessels

Liver - hepatocytes

- bile ducts Urinary bladder - epithelium

Endoatrium Intestinal epithelium Respiratory tract epithelium

Mesothelium

Mammary gland epithelium

Gallbladder epithelium Eidermis (including epid. planta

Epithelium of the tongue, esophagus

Cow Ligament Muscle Blood Vessels

Epidermis

Liver - hepatocytes

- bile ducts

Intestinal epithelium

Mammary gland epithelium

Bladder epithelium; Sweat gland epithelium; Pancreatic epithelium

282,048

Type of medium filament Reaction

IN

In D

N + G N + G

H + 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>9ø10>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 ) +

To - - +

K - - ' ¹ +

K — ” — +

- 7 Table δ. 2 - continued

Type Woven Type of filament 262,046 Reactions Hamster Connective tissue In Muscle D Vessels D + In Lymphoid tissue In Epidermia To (but not Κ,,θ: > - Liver - hepatocytes To including Κ^θ + - bile ducts To _ n _ + Intestinal epithelium To __ n _ + Rat Connective tissue in Muscle D Vessels D + in Liver - hepatocytes To (including Κ _1θ ) + - bile ducts To _ M _ + Intestinal epithelium To o. « _ + Mammary gland epithelium To + Epithelial bladder To tt _ + We Connective tissue in — , Muscle D - Vessels D + In - Liver - hepatocytes To (including K ₁₈ ) + - bile ducts To _ M __ + Mammary gland epithelium To +

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

- 8 Example 3: 262 04β

With regard to the possibility of practical use in the differential diagnosis of tumors, the reactivity of the C-C4 antibody was tested on tissue sections prepared from various human tumors using the immunofluorescence and immunoperoxidase method (see description of the method in Example 2). The results obtained are summarized in Table 3.

Table No. 3 Reactivity of the C-04 antibody on tissue sections of human tumors (+ positive reaction, - negativity,

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

Tumor type Intermediate filament type Reaction

Sarcomas V (or V + D) - Melanomas In - Lymphomas In - Gliomas G - Carcinomas intestine ^To 8,18,19 + breast ^To 7,8,18,19 + lungs ^,8,18,19 + stomach ^To 8,18,19 + ovaries ^To 7,8,18,19 + bladder ^4,7,8,13,17,18,19 + Hepatomas ^To 8.18 +

The results of determining the reactivity of the C-04 antibody with various human tumors confirm the specificity of this antibody for tumors of epithelial origin that contain keratin 18. Together with the negative results in other tumor types, this indicates the diagnostic value of the tested antibody in distinguishing various tumor lesions.

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

The monoclonal antibody produced by the hybridoma RICE0-C-04 _f reacts with keratin from a number of animal species and is useful in various tests including immunohistochemistry on tissue sections, immunoblotting, enzyme-immunological and radioimmunological assays and immunofluorescence, e.g. on cultured cells. The C-04 antibody is therefore useful for detecting cellular cytoskeletal structures containing keratin 18 in humans, but also in various farm and experimental animals. It will find application mainly in routine differential diagnostics in departments of pathological anatomy and oncology, as an important aid to distinguish positively reacting adenocarcinomas from other types of tumors, such as sarcomas, melanomas or lymphomas. It also provides the possibility of distinguishing adenocarcinomas from carcinomas of stratified squamous epithelium that does not contain keratin 18 (e.g. epidermis). Due to its properties, the human medical practice pub can also be used in diagnostics in the field of veterinary medicine, as well as in a number of applications in animal cell biology research, in the departments of healthcare, education, the Czechoslovak Academy of Sciences and the Slovak Academy of Sciences, or 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.