WO1990000404A1 - Enrichissement d'anticorps polyclonaux specifiques a un antigene par la selection de cellules hybrides immortelles - Google Patents

Enrichissement d'anticorps polyclonaux specifiques a un antigene par la selection de cellules hybrides immortelles Download PDF

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Publication number
WO1990000404A1
WO1990000404A1 PCT/US1989/003062 US8903062W WO9000404A1 WO 1990000404 A1 WO1990000404 A1 WO 1990000404A1 US 8903062 W US8903062 W US 8903062W WO 9000404 A1 WO9000404 A1 WO 9000404A1
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WIPO (PCT)
Prior art keywords
antigen
cells
antibody
mixture
sample
Prior art date
Application number
PCT/US1989/003062
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English (en)
Inventor
Joseph C. Fu
Wuan Lu
K. H. Lee
Original Assignee
United Biotech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by United Biotech, Inc. filed Critical United Biotech, Inc.
Publication of WO1990000404A1 publication Critical patent/WO1990000404A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the invention relates to the field of antibody preparation for use in im unoassay procedures . Specifically, the invention relates to the production of high titer polyclonal compositions useful in such assays.
  • the literature relating to the use of antibodies or immunoreactive fragments of the antibodies for immunoassay procedures is so extensive as to- defy enumera ⁇ tion.
  • a variety of techniques have been disclosed for sample preparation, labeling systems, and protocols.
  • the assays can be performed in microtiter wells, supported on membranes, or conducted in test tubes.
  • the protocols may involve direct assay of an analyte, or may involve competition of an analyte for the same immunological re ⁇ agent.
  • Labels may include radioisotopes, enzymes, or fluorescent or colored dye.
  • the initial immunoassay procedures were performed with antibody mixtures obtained as antisera of animals immunized with the relevant antigen.
  • These anti ⁇ bodies are, of course, polyclonal as they are produced by the progeny of numerous stimulated immunocompetent cells, and they react in varying degrees to one or more epitopes associated with the antigen.
  • the cells obtained in the fusion are cultured in a selection medium in which neither the immortalizing cells, i.e., the myeloma cells, nor the antibody-secreting cells can survive alone for extended times or repeated transfers.
  • the antibody-secreting cells would ordinarily die after a few passages; the myeloma cells used in the fusion are chosen so that they lack certain metabolic pathways provided by the antibody-secreting cells, which permit survival on the selected medium. Therefore, the surviving culture will contain only fused cells .
  • the surviving culture is diluted so that individual cultures containing only descendants from an initial par ⁇ ent are obtained.
  • the individual cultures, or clones, are then screened for the desired specificity and affinity and the individual cultures of the correct specificity and ' affinity are used, theoretically in perpetuity, as a source for the desired immunoglobulin.
  • the invention provides a dependable and re ⁇ producible source of polyclonal compositions useful in immunoassay procedures.
  • the compositions of the invention provide higher titers and more reproducible results than the polyclonal compositions obtained from serum, and do not require the tedious screening procedures needed to obtain suitable monoclonal preparations. The necessity to screen individual clones of desired specificity has been shown herein to be unnecessary.
  • the invention is directed to a process for preparing a polyclonal antibody composition which process comprises culturing a mixture of immortalized cells wherein the mixture is capable of secreting antibodies of the desired specificity, and re ⁇ covering the secreted antibodies from the culture.
  • the mixture of cells is preenriched for immortalized cells which secrete antibod ⁇ ies of the desired specificity.
  • a convenient embodiment of this enrichment procedure comprises segregating the desired antibody-secreting cells from undesirable fused cells in the mixture by adhesion of the cells to antigen- coated surfaces.
  • the invention further includes purifica ⁇ tion of the secreted polyclonal mixture from culture fluid, in particular, from ascites fluid obtained when the immortalized cell mixture is injected into suitable mam ⁇ malian subjects, in particular, into mice.
  • the invention relates to polyclonal compositions prepared by the method of the invention, to ascites fluid containing a polyclonal anti ⁇ body mixture, and to methods of immunoassay using the polyclonal compositions of the invention or compositions containing immunoreactive fragments of the polyclonal antibodies contained in the composition.
  • Figure 1 shows two representative comparisons of the titers of the compositions of the invention and antiserum in binding the antigen alpha-hCG.
  • the invention provides polyclonal compositions which may be employed in the known methods for analysis using specific interaction of immunoglobulins with their antigen counterparts.
  • a variety of protocols ranging from direct assays to competitive assays, sandwich assays, and assays dependent on agglutination or distortion of conformation may be employed.
  • the variety of protocols presently used is extensive, but exemplary are the follow ⁇ ing wherein the reagent specifically reactive with analyte will be the invention polyclonal composition.
  • analyte antigen a common protocol is to provide labeled antigen to compete with that in the sample of a second antibody which either reacts with a different antigenic determinant on the antigen or which reacts with the antibody used in the original complexation. The resulting precipitate is then separated from the liquid phase and redissolved.
  • an antibody to the analyte antigen is provided with a label and is used to form the original immune complex which can then be further insolubilized by addition of another antibody.
  • the label is carried by this second antibody rather than the primary complexing immunoglobulin.
  • the foregoing can be (and generally is) modified by adsorbing or covalently attaching one of the reaction components to a solid support and obtaining a specific complex attached to this support.
  • an anti ⁇ body specific to an antigen analyte can be adsorbed, treated with the sample to be analyzed, and then with another antibody reactive to the antigen.
  • the label may be carried either by a competitor to the analyte or by the second antibody layer.
  • the polyclonal compositions of the invention may be used as mixtures of whole antibodies, or the mixture may be fragmented using pepsin or trypsin with or without the presence of reducing agent to produce immunoreactive fragments . It is understood that the polyclonal compositions of the inven ⁇ tion include both those having intact antibodies and those which contain their fragments .
  • a mixture of immortalized cells which secrete antibodies of appropriate specificity and affinity are employed. This mixture is obtained by fusing antibody-secreting cells from an immunized subject with an immortalizing cell, or by immortalizing these antibody- secreting cells by alternative means such as transforma ⁇ tion with virus. The mixture of antibody-secreting cells is then cultured to obtain the polyclonal composition, or is first enriched for cells producing the desired antibody by treating the mixture with a surface coated with antigen to adsorb the desired cellular components of the mixture.
  • the degree of purity required for a particular assay can be variable depending on the nature of the assay and the circumstances. In some instances, as described below, ascites fluid or culture medium contain ⁇ ing the antibodies can be used directly, in other cases purification of the immunoglobulin contained is desirable.
  • the mixture of immortalized cells is prepared by immunizing a suitable subject, such as a mouse, rat or larger animal, with a preparation containing the antigen, and monitoring the subject for antibody titers by standard immunoassay techniques performed on the serum. Standard ELISA techniques are convenient, for example.
  • the animal is optionally boosted with an additional administration of the antigen and antibody-producing cells are recovered.
  • peripheral blood lymphocytes may also be used; this may be preferable in the case of larger animals as sacrifice is then not required.
  • mice Female Balb/C mice, 4-6 weeks old are administered 25 mg of the desired antigen emulsified in Freund's complete adjuvant; (50/50, v/v) simultaneously. The mice are then immunized with the same amount of antigen at 2 week intervals, except emulsified in incomplete Freund's adjuvant. At the end of the fifth week, serum titers of the mice are determined by immunoassay and the animal with highest antibody titer against the antigen is chosen as the source of antibody- secreting cells .
  • the mouse is then injected with 100 mg of antigen in 100 ul phosphate buffered saline (PBS) through the tail vein, and 3 days later the mouse is sacrificed. The spleen is excised and the cells separated.
  • PBS phosphate buffered saline
  • the spleen cells are immortalized using techniques generally known.
  • the most dependable technique is fusion using a fusion enhancer such as polyethylene glycol, but other techniques for immortalization are known, including infection with virus and transformation with DNA which is capable of conferring immortality.
  • spleen cells are fused with about 2 x 107 murine myeloma cells (NS-1, for example) using the protocol described in Oi, V.T., et al "Selected Methods in Cellular Immunology” (1900) p. 351.
  • the resulting hybrid cells are cloned as a group in a flask with liquid selection medium, wherein the medium is chosen so as to be incapable of sup ⁇ porting the growth of unfused immortalizing cells.
  • Com ⁇ monly used myeloma cell lines utilize nucleic acid synthesis pathways which are incompatible with "HAT" (hypoxanthine-aminopterin-thymidine) medium.
  • a suitable medium contains fetal calf serum, lipopolysaccharide (LPS) thymocytes as feeder cells, and HAT. Five days after culturing only the fused cells remain viable.
  • the supernatant from the mixed culture is then verified to contain the desired antibodies using a convenient assay procedure such as ELISA or radioimmunoassay.
  • the mixture of cells is then recovered from the selective medium, and can be subsequently cultured to provide a source of the antibody mixture.
  • the harvested cells can be cultured in vitro using standard media or can be cultured by injection and production of ascites fluid.
  • culturing techniques can be performed directly on this mixture, or the mixture can first be enriched for cells which secrete antibody capable of bind ⁇ ing to the selected antigen as described below, or stimulated for antibody production by transformation, also as described.
  • the culturing may be in vitro or by in vivo production of ascites fluid.
  • Techniques for in vitro culture are well understood in the art, the cells are grown on suitable medium in convenient containers.
  • In vivo techniques are also available, wherein the antibodies are produced in ascites fluid.
  • the subject animal is first primed with 0.5 ml of pristane (2,6,10,14- tetramethyl pentadecane) and the harvested cells are injected into the peritoneal cavity of suitable subjects, preferably syngenic or semi-syngenic mice. This results in formation of antibody-producing tumors , which in turn results in a high concentration of antibody in the peritoneal exudate or ascites fluid of the host mouse.
  • the ascites fluid can be used directly, or if desired, the antibodies can be further purified using standard procedures, such as those of Heide, K., et al, "Handbook of Experimental Immunology” (1978), Weir, D.M., 3rd ed., Blackwell, Oxford, Chapter 7; Fahey, J.L., et al (ibid) , Chapter 8.
  • the supernatant- containing antibody from in vitro culture of the mixture of cells can also be purified.
  • the mixture can first be enriched by adsorption onto an antigen-coated surface. Any convenient container can be used, but a microtiter plate is convenient.
  • the harvested mixture of cells is placed in contact with the antigen- coated surface and allowed to incubate for an effective time period to effect adsorption of the cells secreting the desired antibody immunoreactive with the antigen coat.
  • the surface is then washed to remove unreacted hybridomas and then fresh medium added to cultivate the adsorbed cells.
  • the resulting cultures, which are now enriched for antibody-secreting hybridomas are cultured using in vitro or in vivo methods, as described above, for the unenriched mixture.
  • PBS is added to each well of a microtiter plate. After incubation overnight at -4 C, the solution is remov i, and the nonspecific binding sites blocked by 1% BSA in PBS containing 10 x penicillin/streptomycin and fugiazone at room temperature. After 1 hour, the blocking solution is removed by suction, and the plate is dried and sterilized under UV " light for later use.
  • the cultures obtained in the microtiter wells are then further cultured as described above. If desired, favored wells which contain cells secreting high levels of - ⁇
  • the desired antibody as assessed by a suitable immunoassay procedure, can be chosen for culturing.
  • the fused cells can be stimulated using viral transformation.
  • Eppstein-Barr virus is frequently used, as described by Campbell, A.M., Mabs Technology in “Laboratory Techniques in Biochem & Mol Biol", Berdon, R.H. et al (ed) Elsevier.
  • the production of the desired antibody can be monitored using any convenient immunoassay procedure for specificity and affinity to the desired antigen.
  • the antigen is labeled with I (10 ug/mCi) according to the chloramine T method of Greenwood, F.C., et al, Biochem J (1963) 8 , 9:114.
  • I 10 ug/mCi
  • the solution is centrifuged at 1,500 x g for 20 minutes, and the super ⁇ natant removed. The radioactivity of the pellet is then counted by an LKB minigamma counter.
  • the foregoing assay method is typical, but any convenient method of measuring antibody titer can be used.
  • Example 1 The following examples are intended to il ⁇ lustrate, but not to limit, the invention.
  • Example 1 The following examples are intended to il ⁇ lustrate, but not to limit, the invention.
  • mice Male Balb/C mice, 4-6 weeks old, were immunized subcutaneously with 25 ug alpha-hCG emulsified in Complete Freund's adjuvant. The mice were immunized with the same amount of antigen at 2-week intervals, except that the antigen was emulsified in Incomplete Freund's adjuvant. Seven days after the second injection, serum titers were checked by the RIA assay described above. The mouse with the highest antibody titer (1,800) against alpha-hCG was chosen as a source of antibody-secreting cells for fusion. The selected animal was administered 100 ug alpha-hCG in 100 ul PBS through the tail vein, and the animal sacrificed 3 days later.
  • the spleen cells were harvested, dissociated, and fused with the mouse myeloma cell line NS-1 using standard fusion techniques mediated by PEG.
  • the fusion mixture was harvested and cultured in a flask using HAT selection medium. After 5 days of culture, the supernatant was verified to contain antibodies reactive with alpha-hCG using the RIA assay above.
  • the cells were harvested and treated as follows.
  • a second fraction was subjected to screening.
  • Microtiter plates were coated with hCG as described above.
  • the behavior of the recovered ascites fluids were tested in a radioimmunoassay and compared with serum from a mouse immunized with the antigen.
  • the results are shown in Figure 1.
  • the titer from the enriched ascites fluid (AF-2) was 15,000; that from the unenriched ascites fluid (AF-1), 4,400; and that from serum, 1,800.
  • the results from a second run of a similar experiment are also shown in Figure 1; in this run, the titers for the AF-2 ascites was 400,000, that from the unenriched AF-1 ascites was 71,000, and that for serum 1,700.
  • the ascites fluids recovered as AF-1 and AF-2 in Example 1 were subjected to purification procedures as described above (ammonium sulfate fractionation followed by DEA Sepharose) .
  • 0.8 mg of immunoglobulin was obtained from AF-2, and 1.8 mg from AF-1.
  • AF-1 and AF-2 were coated in microtiter wells.
  • a monoclonal antibody (T-48) known to be specific to alpha-hCG was used as a positive control .
  • the antibody coatings were at 1 ug per well .
  • the coated wells were used in a standard.
  • the results were measured as optical density of a sample containing 25 mlU/ml hCG relative to the optical density of a blank.
  • the absorbance ratios were obtained at 452 nm for 25 mlU/ml hCG standard, and 0 mlU/ml standard.
  • the results were as follows: T-48, 3.49; AF-1, 1.27; AF-2, 2.13.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

On a mis au point des procédés de préparation de compositions d'anticorps polyclonaux de titres élevés et ayant une spécificité voulue destinés à être utilisés dans des procédures de diagnostic par dosage immunologique. Ces procédés emploient des cellules immortalisées cultivées en groupe ou enrichies, par exemple par adsorption sur une matrice de support solide spécifique, et/ou stimulées pour permettre une prolifération par transformation, en groupe pour ensuite être cultivée. Les compositions ainsi obtenues ont une spécificité et un titre supérieurs à celui de l'antisérum polyclonal.
PCT/US1989/003062 1988-07-15 1989-07-17 Enrichissement d'anticorps polyclonaux specifiques a un antigene par la selection de cellules hybrides immortelles WO1990000404A1 (fr)

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US21932188A 1988-07-15 1988-07-15
US219,321 1988-07-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807415B2 (en) * 2005-08-23 2010-10-05 Iq Therapeutics Bv Methods of producing stable B-lymphocytes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
D. CRAWFORD, "Human Hybridomas and Monoclonal Antibodies", published 1985, by Plenum Press (New York, USA); see chapter 3, pages 37 to 53. *
J. GODING, "Monoclonal Antibodies: Principles and Practice", published 1986, by Academic Press (USA); see chapter 5, pages 142 to 218. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807415B2 (en) * 2005-08-23 2010-10-05 Iq Therapeutics Bv Methods of producing stable B-lymphocytes

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