LU85581A1 - MONOCLONAL ANTIBODY SETS AGAINST HUMAN LACTOFERRIN AND HUMAN LYSOZYME - Google Patents
MONOCLONAL ANTIBODY SETS AGAINST HUMAN LACTOFERRIN AND HUMAN LYSOZYME Download PDFInfo
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- LU85581A1 LU85581A1 LU85581A LU85581A LU85581A1 LU 85581 A1 LU85581 A1 LU 85581A1 LU 85581 A LU85581 A LU 85581A LU 85581 A LU85581 A LU 85581A LU 85581 A1 LU85581 A1 LU 85581A1
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- Prior art keywords
- lactoferrin
- lysozyme
- monoclonal antibodies
- mice
- human milk
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- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 title description 5
- 102000050459 human LTF Human genes 0.000 title description 5
- 101001018100 Homo sapiens Lysozyme C Proteins 0.000 title description 4
- 102000010445 Lactoferrin Human genes 0.000 claims description 18
- 108010063045 Lactoferrin Proteins 0.000 claims description 18
- 108010014251 Muramidase Proteins 0.000 claims description 18
- 102000016943 Muramidase Human genes 0.000 claims description 18
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 18
- 235000021242 lactoferrin Nutrition 0.000 claims description 18
- 229940078795 lactoferrin Drugs 0.000 claims description 18
- 235000010335 lysozyme Nutrition 0.000 claims description 18
- 239000004325 lysozyme Substances 0.000 claims description 18
- 229960000274 lysozyme Drugs 0.000 claims description 18
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims description 17
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- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Rehabilitation Therapy (AREA)
- Rheumatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
kk
Ensembles d'anticorps monoclonaux dirigés contre la lactoferrine humaine et le lysozyme humainSets of monoclonal antibodies to human lactoferrin and human lysozyme
La présente invention concerne des ensembles d1 anticorps monoclonaux dirigés respectivement contre la lactoferrine humaine et contre le lysozyme humain.The present invention relates to sets of monoclonal antibodies directed respectively against human lactoferrin and against human lysozyme.
5 Lactoferrine et lysozyme :5 Lactoferrin and lysozyme:
La lactoferrine et le lysozyme se retrouvent dans de nombreuses sécrétions telles que le lait, la salive, les larmes, les fluides pancréatiques, 1*urine, la bile et les fluides synoviaux. Leur présence a été détectée dans de nom-10 breux tissus parmi lesquels nous citerons les poumons, les cellules gastriques et duodenales, le tractus génital et les yeux. (S.A.C. Sykes, M.J. Thomas, D.J. Goldie et G.M.Lactoferrin and lysozyme are found in many secretions such as milk, saliva, tears, pancreatic fluids, urine, bile and synovial fluids. Their presence has been detected in many tissues, among which we will mention the lungs, gastric and duodenal cells, the genital tract and the eyes. (S.A.C. Sykes, M.J. Thomas, D.J. Goldie and G.M.
Turner, Clin. Chim. Acta 122 (1982) 385-393, T.L. Peeters, Y.R. Depraetere et G.R. Vantrappen, Clin. Chem. 24 (1978) 15 2155-2157, R.M. Bennett et J.L. Skosey, Arthritis and Rheu- matism 20 (1977) 84-90). L'association des deux antigènes a été décrite notamment dans les granules de leucocytes polymorphonucléaires (M.S. Lefell et J.K. Spitznagel Infect. Immun. 6 (1972) 1761).Turner, Clin. Chim. Acta 122 (1982) 385-393, T.L. Peeters, Y.R. Depraetere and G.R. Vantrappen, Clin. Chem. 24 (1978) 15 2155-2157, R.M. Bennett and J.L. Skosey, Arthritis and Rheumatism 20 (1977) 84-90). The combination of the two antigens has been described in particular in polymorphonuclear leukocyte granules (M.S. Lefell and J.K. Spitznagel Infect. Immun. 6 (1972) 1761).
20 4 Lactoferrine :20 4 Lactoferrin:
La lactoferrine est une glycoprotéine de 70.000 daltons (poids moléculaire). Elle est synthétisée dans les leucocytes polymorphonucléaires neutrophiles et le lait.Lactoferrin is a glycoprotein of 70,000 daltons (molecular weight). It is synthesized in polymorphonuclear neutrophil leukocytes and milk.
25 Elle exerce des propriétés bactériostatiques (Bullen C.L. et25 It has bacteriostatic properties (Bullen C.L. and
Willie A.T,, Br. Med. J. 3 (1971) 338). Le taux de lactoferrine l - 2 - est modifié au cours d'infections (J. Breton-Gorius, D.Y.Willie A.T ,, Br. Med. J. 3 (1971) 338). The level of lactoferrin l - 2 - is modified during infections (J. Breton-Gorius, D.Y.
Kasson, B. Burict, J.L. Vilde et C. Griscelli, J. Pedia-trics 94 (1979) 1 - 8), au cours de maladies pancréatiques (S.S. Fedail, P.R. Salmon, R.F. Harvey, A.E. Read, The 5 Lancet 28 (1978) 181-182), Le taux de lactoferrine est également perturbé dans certaines maladies inflammatoires et il reflète ”1*activité inflammatoire" (R,K. Bennett et S.L. Skosey, Arthritis and Rheumatism 20 (1977) 84-90).Kasson, B. Burict, JL Vilde and C. Griscelli, J. Pedia-trics 94 (1979) 1 - 8), in pancreatic diseases (SS Fedail, PR Salmon, RF Harvey, AE Read, The 5 Lancet 28 ( 1978) 181-182), The level of lactoferrin is also disturbed in certain inflammatory diseases and it reflects "1 * inflammatory activity" (R, K. Bennett and SL Skosey, Arthritis and Rheumatism 20 (1977) 84-90).
« i ; 10 Lysozyme :"I; 10 Lysozyme:
Le lysozyme est une protéine de faible poids moléculaire (14.000 daltons). Il est produit en quantités relativement importantes dans les cellules phagocytaires, les leucocytes polymorphonucléaires, les macrophages. C'est un 15 catalyseur puissant de la destruction de certaines parois bactériennes et il pourrait intervenir dans l'immunité non spécifique, La production de lysozyme varie dans la leucémie et la sarcoïdose, dans des affections gastrointestinales, rénales et urinaires et dans le rejet de 20 greffes (M.J, Thomas, A, Russo, P. Craswell, M. Ward et I. Steinhardt, Clin. Chem. 27 (1981) 1223-1226).Lysozyme is a low molecular weight protein (14,000 daltons). It is produced in relatively large quantities in phagocytic cells, polymorphonuclear leukocytes, macrophages. It is a powerful catalyst for the destruction of certain bacterial walls and it could intervene in non-specific immunity. The production of lysozyme varies in leukemia and sarcoidosis, in gastrointestinal, renal and urinary disorders and in the rejection of 20 transplants (MJ, Thomas, A, Russo, P. Craswell, M. Ward and I. Steinhardt, Clin. Chem. 27 (1981) 1223-1226).
Dans certains cas, le dosage indépendant des deux protéines, la lactoferrine et le lysozyme, présente un intérêt du fait que les deux protéines n'ont pas exactement 25 la même localisation et que la mesure de leur rapport peut „aider, à établir un diagnostic.In some cases, the independent assay of the two proteins, lactoferrin and lysozyme, is of interest because the two proteins do not have exactly the same location and the measurement of their ratio can help to establish a diagnosis. .
IBien que l'on puisse obtenir facilement des anticorps polyclonaux contre l'une ou l'autre protéine, les méthodes de dosage utilisant de tels anticorps manquent de précision.While polyclonal antibodies to one protein or another can be readily obtained, assay methods using such antibodies lack precision.
30 Dès lors, l'obtention d'anticorps monoclonaux spécifiques du lysozyme et de la lactoferrine permettrait la mise au point de systèmes de dosage plus sensibles de ces antigènes30 Therefore, obtaining monoclonal antibodies specific for lysozyme and lactoferrin would allow the development of more sensitive assay systems for these antigens.
Iet conduirait à une amélioration de la détection des nombreuses affections mentionnées précédemment.It would lead to improved detection of the many aforementioned conditions.
33 à33 to
l Il I
- 3 - L’invention concerne des ensembles d’anticorps monoclonaux dirigés contre des protéines de lait humain purifiées, respectivement contre la lactoferrine et le lysozyme.The invention relates to sets of monoclonal antibodies directed against purified human milk proteins, respectively against lactoferrin and lysozyme.
55
Préparation :Preparation:
La préparation des anticorps monoclonaux s’effectue selon une technique de fusion cellulaire décrite par Köhler G. et Milstein C. (Eur. J. ïmmunol. 6 (1975)» 511 et Nature 10 256 (1975) 495-497 et modifiée par Franssen J.D., Hérion P.The preparation of the monoclonal antibodies is carried out according to a cell fusion technique described by Köhler G. and Milstein C. (Eur. J. immunol. 6 (1975) "511 and Nature 10 256 (1975) 495-497 and modified by Franssen JD, Hérion P.
et Urbain J. (Protides of Biol. Fluids 29 (1981) 645, Ed. Peeters, Pergamon Press).and Urbain J. (Protides of Biol. Fluids 29 (1981) 645, Ed. Peeters, Pergamon Press).
La présente invention couvre également l’application, 15 pour la production des ensembles d’anticorps monoclonaux précités, du procédé caractérisé par l’immunisation de souris au moyen de l’antigène - soit la lactoferrine de lait humain, soit le lysozyme de lait humain, par la fusion cellulaire des cellules de rate de souris immunisée soit avec la 20 lactoferrine, soit avec le lysozyme avec des cellules de myélome de souris sélectionnées, telles que des plasmacytomes de souris BALB/c non sécréteurs, par la sélection des hybridomes sécréteurs d’anticorps spécifiques, soit de la lactoferrine, soit du lysozyme, par le clonage de ces hybridomes, par la 25 production in vivo des anticorps monoclonaux des deux spécificités en ascite, par l’identification des anticorps sécrétés par ces clones et par la caractérisation des classes d’immunoglobulines de ces anticorps, par l’étude de l’affinité relative des anticorps monoclonaux et de leur spécificité épi-30 topique.The present invention also covers the application, for the production of the above-mentioned sets of monoclonal antibodies, of the method characterized by the immunization of mice by means of the antigen - either human milk lactoferrin or human milk lysozyme , by cell fusion of spleen cells from mice immunized either with lactoferrin or with lysozyme with selected mouse myeloma cells, such as non-secretory BALB / c mouse plasmacytomas, by selection of secretory hybridomas from d specific antibodies, either lactoferrin or lysozyme, by the cloning of these hybridomas, by the in vivo production of monoclonal antibodies of the two specificities in ascites, by the identification of the antibodies secreted by these clones and by the characterization of classes of immunoglobulins of these antibodies, by studying the relative affinity of monoclonal antibodies and their epi-topical specificity.
L’application du procédé susdit est décrite, à titre non limitatif, dans l’exemple suivant : 35 | *“ ’ 1. Immunisation des sourisThe application of the above process is described, without limitation, in the following example: 35 | * "’ 1. Immunization of mice
Les antigènes ont été purifiés à partir de lait humain provenant de donneuses saines et leur homogénéité a été testée par électrophorèse sur gel d’acrylamide.The antigens were purified from human milk from healthy donors and tested for homogeneity by acrylamide gel electrophoresis.
5 Les préparations d’antigènes purifiés ont été utilisées pour immuniser des souris femelles (souches BALB/c) âgées de 8 semaines.5 The purified antigen preparations were used to immunize 8 week old female mice (BALB / c strains).
On immunise d’abord les souris avec 200 ug d’antigène émulsifié dans l’adjuvant complet de Freund et on 10 injecte le mélange par moitié par voie sous-cutanée en points multiples dans le dos et par moitié par voie intra-péritonéale. Deux semaines plus tard, les souris reçoivent une injection de rappel identique. Après une période de repos de 1 mois, les souris reçoivent au 15 jour moins quatre avant la fusion, une injection de 20 μg d'antigène (dans du NaCl 0,15 M) par voie intraveineuse.The mice are first immunized with 200 µg of emulsified antigen in Freund's complete adjuvant and the mixture is injected in half by subcutaneous route at multiple points in the back and in half by intraperitoneal route. Two weeks later, the mice receive an identical booster injection. After a rest period of 1 month, the mice receive, at 15 days minus four before fusion, an injection of 20 μg of antigen (in 0.15 M NaCl) intravenously.
Au jour zéro, on sacrifie les souris et on prélève la rate pour la préparation des cellules destinées aux 20 fusions cellulaires.On day zero, the mice are sacrificed and the spleen is removed for the preparation of cells for cell fusion.
2. Cellules de myélome de souris2. Mouse myeloma cells
La méthode décrite par Franssen J.D., Hérion P. et Urbain J. (Protides of Biol. Fluids 29 (1981) 645, 25 Ed. Peeters, Pergamon Press) a-été utilisée.The method described by Franssen J.D., Hérion P. and Urbain J. (Protides of Biol. Fluids 29 (1981) 645, 25 Ed. Peeters, Pergamon Press) was used.
On a sélectionné des cellules de myélome de souris possédant les propriétés suivantes : croissance rapide in vivo et in vitro, capacité de pouvoir être clonées sans le support de cellules nourricières et en milieu 30 semi-solide, capacité de fusionner à taux élevé. Cette lignée a été développée par Shulman M., Wilde C.D. et Köhler G. (1978) Nature 276, 269-270.Mouse myeloma cells were selected having the following properties: rapid growth in vivo and in vitro, ability to be cloned without the support of feeder cells and in semi-solid medium, ability to merge at high rate. This line was developed by Shulman M., Wilde C.D. and Köhler G. (1978) Nature 276, 269-270.
Dans ce but, des cellules myélomateuses de souris, telles que des plasmacytomes de souris BALB/c non sécréteurs, ont ^ été clonées en agar. Les cellules capables de pousser dans ces conditions ont alors été reclonées en agar puis injec- - 5 - tees uar voie intrapéritonéale dans des souris syngé-niques.For this purpose, mouse myeloma cells, such as non-secretory BALB / c mouse plasmacytomas, were cloned into agar. The cells capable of growing under these conditions were then recloned into agar and then injected via the intraperitoneal route into syngeic mice.
Après une à deux semaines, les cellules myélomateuses Be développent sous forme de tumeur dans les souris 5 injectées. Les cellules sont alors collectées dans la cavité péritonéale, resélectionnées, reclonées en agar et essayées dans une expérience de fusion cellulaire.After one to two weeks, the Be myeloma cells develop as a tumor in the injected mice. The cells are then collected in the peritoneal cavity, reselected, recloned in agar and tested in a cell fusion experiment.
Le cycle décrit ci-dessus est recommencé trois fois.The cycle described above is repeated three times.
A 1*issue du procédé, on obtient des sous-clones 10 sélectionnés de la lignée myélomateuse, SP 2/0 - Ag 14, possédant les propriétés voulues pour leur fusion cellulaire ultérieure.At the end of the process, selected subclones 10 of the myeloma line, SP 2/0 - Ag 14, are obtained which have the properties desired for their subsequent cell fusion.
3. Fusion cellulaire ' 15 Pour effectuer les fusions, on a utilisé les matières et milieux suivants. On a obtenu chez GIBCO le milieu DMEM que l’on a complété avec du sérum de cheval (10 90, du sérum de veau (5 90» des acides aminés non essentiels, du pyruvate de sodium (1 mM), de la pénicilline (100 U/ml) 20 et de la streptomycine (100 μg/ml).3. Cell Fusion To perform the fusions, the following materials and media were used. DMEM medium was obtained from GIBCO, which was supplemented with horse serum (10 90, calf serum (5 90% non-essential amino acids, sodium pyruvate (1 mM), penicillin ( 100 U / ml) 20 and streptomycin (100 μg / ml).
Le milieu de sélection complet (HAT) se composait du milieu décrit ci-dessus ainsi que d»hypoxanthine (10-4 M), d*aminoptérine (4.10*"^) et de thymidine (1.5.1O""'* M).The complete selection medium (HAT) consisted of the medium described above as well as hypoxanthine (10-4 M), aminopterin (4.10 * "^) and thymidine (1.5.1O" "'* M) .
Le milieu solide contenant l’agar (1,5 90 comportait 25 les ingrédients décrits ci-dessus. Les cellules ont été cultivées à 37°C dans une atmosphère humide de 5 % C0£ et 95 % d’air.The solid medium containing the agar (1.590 contained the ingredients described above. The cells were cultured at 37 ° C. in a humid atmosphere of 5% CO 2 and 95% air.
La fusion avec des cellules provenant d’une lignée cellulaire de myélome de souris (sous-clones sélection-30 nés SP 2/0 - Ag 14) a été réalisée selon le procédé deThe fusion with cells from a mouse myeloma cell line (selection subclones-30 born SP 2/0 - Ag 14) was carried out according to the method of
Franssen J.D., Hérion P. et Urbain J. (1981), Proc.Franssen J.D., Hérion P. and Urbain J. (1981), Proc.
XXIX Colloquium on Protides of the Biological Fluids, Peeters Ed.XXIX Colloquium on Protides of the Biological Fluids, Peeters Ed.
Selon ce protocole, 3.5 10 cellules de rate de souris J 35 ont été fusionnées avec 3.10^ cellules de myélome deAccording to this protocol, 3.5 10 J 35 mouse spleen cells were fused with 3.10 ^ myeloma cells.
I II I
! - 6 - souris de la lignée SP 2/0 - Ag 14· Pour chaque fusion, on distribue les cellules fusionnées dans des plaques de microculture à 96 puits en effectuant des dilutions. Les cultures sont régulièrement alimentées par du 5 milieu frais. Au Jour 15 après la fusion, 50 μΐ de surnageant de chaque culture est testé dans un immuno essai en phase solide pour détecter la sécrétion d' anticorps.! - 6 - mice of the SP 2/0 line - Ag 14 · For each fusion, the fused cells are distributed in 96-well microculture plates by making dilutions. The cultures are regularly fed with fresh medium. On Day 15 after the fusion, 50 μΐ of supernatant from each culture is tested in an immunoassay in solid phase to detect the secretion of antibodies.
10 4. Sélection dfhybridomes sécréteurs d1anticorps spécifiques10 4. Selection of hybridomas secreting specific antibodies
Les anticorps spécifiques de la lactoferrine et les anticorps spécifiques du lysozyme sont détectés par 15 le test ELISA MEnzyme-linked Immunosorbent assay" suivant î 50 ul d’une solution d’antigène (2 ug/ml de lactoferrine humaine purifiée ou 2 ug/ml de lysozyme humain purifié) dans du tampon PBS (0,85 % de NaCl, 0,15 M phosphate) pH 7,4 sont incubés dans des puits 20 de plaques en PVC à 96 puits durant une nuit à 4°C·Lactoferrin specific antibodies and lysozyme specific antibodies are detected by the MEnzyme-linked Immunosorbent assay ELISA according to 50 µl of antigen solution (2 µg / ml purified human lactoferrin or 2 µg / ml purified human lysozyme) in PBS buffer (0.85% NaCl, 0.15 M phosphate) pH 7.4 are incubated in wells of 96-well PVC plates overnight at 4 ° C
Les sites d’absorption restant sont alors saturés par incubation avec 50 μΐ de tampon PBS pH 7,4 contenant 1 % d’albumine bovine. Les plaques sont alors lavées plusieurs fois avec du tampon PBS pH 7,4 contenant 25 0,1 % d*albumine bovine. Les surnageants de culture d*hybridomes ou les dilutions d’antisérum de souris sont mis à réagir avec l’antigène adsorbé dans les cupules durant une nuit à 4°C.The remaining absorption sites are then saturated by incubation with 50 μΐ of PBS buffer pH 7.4 containing 1% bovine albumin. The plates are then washed several times with PBS buffer pH 7.4 containing 0.1% bovine albumin. Culture supernatants of hybridomas or dilutions of mouse antiserum are reacted with the antigen adsorbed in the wells overnight at 4 ° C.
Les anticorps fixés sont alors révélés par des immuno-50 globulines de chèvre anti-immunoglobulines de souris marquées à la peroxydase (O’Sullivan M.J. et Marks V., Methods in Enzymology 75 (1981) 147) en solution à 1 ug par ml dans du tampon PBS pH 7,5 contenant 10 de sérum de cheval, 1 % d’albumine bovine et 0,1 % de 55 Tween 80 (Polyéthylène Sorbitan monooléate, USB, s - 7 -The fixed antibodies are then revealed by goox immuno-globulins anti-mouse immunoglobulin of mouse labeled with peroxidase (O'Sullivan MJ and Marks V., Methods in Enzymology 75 (1981) 147) in solution at 1 ug per ml in PBS buffer pH 7.5 containing 10 horse serum, 1% bovine albumin and 0.1% 55 Tween 80 (Polyethylene Sorbitan monooleate, USB, s - 7 -
Cleveland, Ohio, U.S.A.). L'incubation des réactifs précités se poursuit pendant 4 heures.Cleveland, Ohio, U.S.A.). Incubation of the above reagents continues for 4 hours.
Après plusieurs lavages par du tampon PBS pH 7,5» la peroxydase conjuguée est mise en évidence par addition 5 de 50 ul du substrat chromogène (0,4 mg/ml d'ortho- phénylènediamine contenant 0,2 mg/ml de peroxyde d'urée dans du tampon citrate 0,1 K pH 5).After several washes with PBS buffer pH 7.5 ", the conjugated peroxidase is demonstrated by the addition of 50 μl of the chromogenic substrate (0.4 mg / ml of ortho-phenylenediamine containing 0.2 mg / ml of peroxide). urea in 0.1 K citrate buffer pH 5).
La réaction est arrêtée par addition de 50 ni d'HCl 5N et l'absorbance est lue à 492 nm dans un lecteur auto-10 matique (Dynatech AM 120).The reaction is stopped by adding 50 μl of 5N HCl and the absorbance is read at 492 nm in an automatic reader (Dynatech AM 120).
5. Clonage et identification5. Cloning and identification
Les cellules d'hybridomes donnant une réaction positive soit dans l'enzymoimmunoessai caractéristique de la 15 lactoferrine, soit dans l'enzymoimmunoessai caractéris tique du lysozyme sont propagées puis clonées en agarose selon Franssen J.D, et al (1981) (cf. référence point 2). i Les clones qui sécrètent des immunoglobulines in situ | dans 1'agarose sont retestés par l'enzymoimmunoessai 20 décrit au point 4 pour la sécrétion d'anticorps spéci fiques respectivement de la lactoferrine et du lysozyme et soumis à un nouveau clonage afin d'assurer la monoclonalité. Ce protocole permet de récupérer au moins deux hybridomes distincts sécrétant des anticorps 25 dirigés contre la lactoferrine humaine et au moins deux hybridomes distincts sécrétant des anticorps dirigés contre le lysozyme, 6« Production d'anticorps monoclonaux en ascite 30 2.10° cellules d’hybridomes clonées sont injectées intrapéritonéalement dans des souris (syngéniques ou F1 progeny) qui ont été traitées au pristane (2, 6, 10, 14 tétraméthyl-pentadécane).The hybridoma cells giving a positive reaction either in the enzymoimmunoassay characteristic of lactoferrin or in the enzymoimmunoassay characteristic of lysozyme are propagated and then cloned into agarose according to Franssen JD, et al (1981) (cf. reference point 2 ). i Clones that secrete immunoglobulins in situ | in agarose are retested by the enzymoimmunoassay described in point 4 for the secretion of antibodies specific for lactoferrin and lysozyme respectively and subjected to a new cloning in order to ensure monoclonality. This protocol makes it possible to recover at least two distinct hybridomas secreting antibodies directed against human lactoferrin and at least two distinct hybridomas secreting antibodies directed against lysozyme, 6 “Production of monoclonal antibodies in ascites 30 2.10 ° cloned hybridoma cells are injected intraperitoneally into mice (syngenic or F1 progeny) which have been treated with pristane (2, 6, 10, 14 tetramethyl-pentadecane).
55 I55 I
- 6 - ο 7* Purification des anticorps monoclonaux- 6 - ο 7 * Purification of monoclonal antibodies
Le procédé décrit par Stachelin T., ÏSobbs D.S,, v Hsiang-Fu K., Chu-yen L. et Petska S. (1931)» J. Biol. Chem. 256, 9750-9754, a été suivi dans sa 5 totalité, si ce n'est que la diéthylaminoéthyl (DEAE)- cellulose a été remplacée par la DEAE - Affi-gel Blue (Biorad) pour éliminer les protéases. Les opérations se font à 4°C, Les fractions d’anticorps monoclonaux sont recueillies, précipitées deux fois au sulfate de 10 sodium solide (jusqu'à obtenir une concentration finale de 18 % de ce sel et dialysées contre un tampon carbonate 0,1 M pH 8,5.The process described by Stachelin T., ÏSobbs D.S ,, v Hsiang-Fu K., Chu-yen L. and Petska S. (1931) »J. Biol. Chem. 256, 9750-9754, was followed in its entirety, except that diethylaminoethyl (DEAE) - cellulose was replaced by DEAE - Affi-gel Blue (Biorad) to eliminate the proteases. The operations are carried out at 4 ° C. The monoclonal antibody fractions are collected, precipitated twice with solid sodium sulphate (until a final concentration of 18% of this salt is obtained and dialyzed against a carbonate buffer 0.1 M pH 8.5.
8. Caractéristiques des anticorps monoclonaux s 15 La classe d’immunoglobuline à laquelle appartiennent les anticorps monoclonaux est déterminée par test d’Ouchterlony.8. Characteristics of the monoclonal antibodies The class of immunoglobulin to which the monoclonal antibodies belong is determined by the Ouchterlony test.
Claims (2)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU85581A LU85581A1 (en) | 1984-10-10 | 1984-10-10 | MONOCLONAL ANTIBODY SETS AGAINST HUMAN LACTOFERRIN AND HUMAN LYSOZYME |
| EP85870138A EP0181851A1 (en) | 1984-10-10 | 1985-10-10 | Kits of monoclonal antibodies against human lactoferrin and human lysozyme |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU85581 | 1984-10-10 | ||
| LU85581A LU85581A1 (en) | 1984-10-10 | 1984-10-10 | MONOCLONAL ANTIBODY SETS AGAINST HUMAN LACTOFERRIN AND HUMAN LYSOZYME |
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| LU85581A1 true LU85581A1 (en) | 1986-06-11 |
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| LU85581A LU85581A1 (en) | 1984-10-10 | 1984-10-10 | MONOCLONAL ANTIBODY SETS AGAINST HUMAN LACTOFERRIN AND HUMAN LYSOZYME |
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| WO1992017786A1 (en) * | 1991-04-01 | 1992-10-15 | Sri International | Assay device and method of detecting chitin |
| CN114720358B (en) * | 2022-04-11 | 2022-09-27 | 浙江普罗亭健康科技有限公司 | Antibody combination for substituting side scattered light signals in mass spectrum flow type blood tumor immunology typing and application |
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