WO1983003678A1 - Method of affinity purification employing monoclonal antibodies - Google Patents

Method of affinity purification employing monoclonal antibodies Download PDF

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Publication number
WO1983003678A1
WO1983003678A1 PCT/US1983/000524 US8300524W WO8303678A1 WO 1983003678 A1 WO1983003678 A1 WO 1983003678A1 US 8300524 W US8300524 W US 8300524W WO 8303678 A1 WO8303678 A1 WO 8303678A1
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WO
WIPO (PCT)
Prior art keywords
environment
antigen
antibody
process according
monoclonal antibody
Prior art date
Application number
PCT/US1983/000524
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English (en)
French (fr)
Inventor
Incorporated Hybritech
Richard M. Bartholomew
Daniel E. Beidler
Gary S. David
Original Assignee
Hybritech 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 Hybritech Inc filed Critical Hybritech Inc
Priority to AU15507/83A priority Critical patent/AU1550783A/en
Priority to JP50166683A priority patent/JPS59500720A/ja
Priority to GB08332644A priority patent/GB2128630B/en
Publication of WO1983003678A1 publication Critical patent/WO1983003678A1/en
Priority to DK564983A priority patent/DK564983D0/da
Priority to FI834530A priority patent/FI88403C/fi
Priority to NO834535A priority patent/NO168969C/no
Priority to DK192191A priority patent/DK192191A/da

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies

Definitions

  • This invention relates to the purification of anti gens and antibodies by affinity chromatography. I another aspect it relates to monoclonal antibodies.
  • a pH of less than 3 or greater than 11 or concentrated chaotrope such as guanidine or urea solution can denature the antigen and the antibodies, diminishing if not destroying, the immunochemical and/or biologica properties of the antigen and shortening the useful lif of the immunoadsorbent.
  • immobilized antibodies o low affinity as an immunoadsorbent.
  • Use of these anti ⁇ bodies permits elution of the antigen from the body of immunoadsorbent using mild, non-denaturing conditions.
  • the requisite step of washing the column to elute impurities from the bound antigen also elutes some of the antigen, so much so that the efficiency of separation i greatly reduced.
  • low affinity antibodie cannot efficiently bind antigens which are present in th media at relatively low concentrations, i.e., less tha about 10 ng/ml.
  • hybridomas are -formed by the rando fusion of B-lymphocytes with myeloma cells in the presence of a fusion promoting agent.
  • Each hybridoma of the large population of hybridomas which can be produced by a fusion secretes a different monoclonal antibody.
  • the population of hybridomas is screened to select for furthe cloning those that secrete an antibody of the desired antigenic specificity in order to obtain useful quantities of antibody.
  • hybridomas which can be the product o multiple fusions, and identify those, which produce monoclonal antibody having a high affinity in a firs environment and a low affinity in a second environment an clone at least one of the hybridomas to obtain a suffi cient quantity of the antibody it produces to permi its use as a highly effective immunoadsorbent for affinit chromatography.
  • an antibody is considere to exhibit a high affinity when its affinity constant (Ka is about >_ 10 9 and to exhibit a low affinity when its K is about l ⁇ 8.
  • Figures 1 and 2 are graphs of data reflecting th effect of changes in pH on the desorption of radiolabele human growth hormone bound to four different monoclona antibodies immobilized on a solid phase.
  • purification of a antigen is accomplished by a process comprising th steps: a) selecting a monoclonal antibody having a high affinity for the antigen in a first environment and a low affinity for the antibody in a second environment, neither environment causing substantial, irreversible changes in the desired immunochemical properties of the antigen; b) immobilizing the antibody on a solid support; c) contacting the immobilized antibody with a sample containing impure antigen in the first environment to bind the antigen to the antibody; d) separating unbound impurities from the bound antigen; and e) eluting the antigen in a purified form from the immobilized antibody using, as an eluant, a medium which is the second environment.
  • antibodies useful in our invention can be obtained by screening the antibodies produced by a population of hybridomas obtained by the fusion, using known methods, of myeloma cells with B-lymphocytes.
  • the B—lymphocytes are typically spleen cells taken from a hype immunized animal to which the target antigen has previously been administered as an immunogen.
  • those hybridomas that produce monoclonal antibodies whose specificities are against the desired antigen have been identified, they can be further screened to identify those that produce antibodies whose affinities vary with changes in environment which are not damaging to the antigen or antibody.
  • antigens are usually stable in solution within the pH range of 4-10.5.
  • the population of monoclonal antibodies is screened to iden ⁇ tify those which have a high affinity for the antibody at one pH within the range, i.e., a Ka of about 10 9 and preferably > 10l0 and a low affinity at a second pH,
  • f Q i.e., a Ka of about 10 8 and preferably less than 10 within the same range.
  • This kind of screening can be don by immobilizing the antibody on a solid support and, afte permitting it to bind antigen, measuring the extent o desorption of the antigen that occurs at different p levels, a measurement which can be made, for instance, b employing radiolabeled antigen and counting the radiatio emitted by the solid phase and/or supernatant.
  • a simila screen can be carried out to identify antibodies tha respond to other kinds of environmental change. It is presently preferred to exploit monoclona antibodies whose capability to bind antigen is sensitiv to changes in pH.
  • the antibody is se lected to have a high affinity at one pH and a low af finity at a second pH which may be higher or lower tha the first pH ' .
  • the first pH will be at or near p 7 although it need not be.
  • tb select antibodies which respon to a different kind of change in environmental condition
  • a monoclonal antibody can be selected whic undergoes a change from high low affinity in the presenc of a chaotropic solution as the eluting medium.
  • Amon suitable chaotropes are KBr, KI, KSCN, guanidine, urea an MgCl2»
  • monoclonal antibodies can be selecte having a Ka > 10-** 1 in the absence of chaotrope but whic has a Ka of _ ⁇ 10*** in the presence of the particula chaotrope whose concentration is not detrimented to th antigen in question.
  • the selection for chaotrope sen sitivity can also be made in buffers at a specific pH.
  • antibodies can be selected which are sensitive to changes in pH in the presence of a constant concentration of a chaotrope.
  • Monoclonal antibodies whose affinity for an antigen is adequately lowered by changes other than pH or concen ⁇ tration of chaotrope can also be selected.
  • media sensitivity for which the antibodies can be screened to select those whose antigen binding ability i affected by a change in eluting medium can include borat sensitivity, methylmannoside sensitivity and sensitivit to non-ionic or ionic detergents and reagents whic affect specific amino acids such as tryptophan and tyro sine.
  • a selecte monoclonal antibody can be bound to any of the soli supports commonly used in affinity chromatography.
  • Thes include sepharose, polystyrene, glass, nylon, cellulose polymethyl methacrylate, silicagel, polyacrylamide an nitrocellulose.
  • Example 1 illustrates the application o the present invention to obtaining monoclonal antibodie whose binding affinity for an antigen varies from a hig affinity in a first environment to a low affinity in second environment, neither environment causing damage t the immunochemical properties of the antigen and thei usefulness as immunoadsorbents for affinity chromato graphy.
  • Example 1 illustrates the application o the present invention to obtaining monoclonal antibodie whose binding affinity for an antigen varies from a hig affinity in a first environment to a low affinity in second environment, neither environment causing damage t the immunochemical properties of the antigen and thei usefulness as immunoadsorbents for affinity chromato graphy.
  • HGH human growth hormone
  • NS-1 or SP-2/ lines poly ethylene glycol with mouse myeloma cells
  • the resulting hybridomas were cloned and screene to determine those secreting antibody specific for HGH b a radioimmunoassay employing 125 ⁇ -HGH and horse anti mouse IgG on sepharose beads.
  • the hybridomas producin anti-HGH were further screened to identify those producin antibodies having a Ka of at least about lO-**' at pH 7 These were further screened to identify those whose affin ities were sensitive to changes in pH over the range from to 10.5.
  • the supernatants eluted from antibodies A and C at p 4.0 and 10.5 were added to PBS buffer (10% in horse serum and adjusted to pH 7 and the samples pooled.
  • the poole samples were incubated with polystyrene balls coated wit antibodies A, B, C and D and two other monoclonal anti bodies against HGH. Each of these antibodies recogniz different areas of the HGH molecule.
  • the antibody was bound to sepharose bea using the CNBr technique at a concentration of 1 mg antibody per 1 ml of packed sepharose beads and used purify PAP from seminal fluid as follows. A 170 J ⁇ !
  • the PAP solution was passed through a column con taining 1.5 ml of the sepharose beads at the rate of ml/hour and the column washed with 7.5 mis of the startin buffer. Immunometric assay of the eluant (5 mis of sampl and 7.4 ml of wash liquid) demonstrated that 99.3% of th PAP had adsorbed to the column.
  • the PAP was eluted wit 0.1 M acetate buffer, pH 4 containing 0.15 M NaCl. Thre 1 ml fractions were collected and dlalyzed overnight vs 50 mM citrate, pH 6.0.
  • the PAP content of the pooled an dialyzed fractions was determined by immunoradiometri assay to be 54% of the total applied to the column Purity of the dialyzed material was determined by sodiu dodecyl sulfate and Ornstein-Davis PAGE. A single ban was observed in each case. Enzymatic activity measure ments were done and documented that the purified PA retained its enzymatic activity.
  • the retention of 46% of the PAP on the column i likely the result, at least in part, of non-specifi binding and the use of a large excess of antibody whic results in antigen "trail w from the column.
  • the forme can be reduced by pretreating the column with sample unde the conditions at which elution will be accomplishe followed by extensive washing to remove any materia which will elute. The latter can be reduced by lowerin the . concentration of bound antibody.
  • sepharos is not an ideal matrix for affinity chromatography becaus of the heterogeneity of pore size, resulting in diffusio and steric problems.
  • Hybridomas which produce monoclona antibodies against chlamydial antigen obtained by fusin spleen cells from hyperimmunized Balb/c mice with mous myeloma cells as described in Example 1, were screened fo
  • Table 3 The data in Table 3 were obtained by coating th antigen on microtiter plates and incubating it with solution of each of the antibodies in a buffer at th concentration of the detergent indicated in the tabl After incubation, the plate is washed and reacted wi polyclonal sheep anti-mouse antibodies labeled wi horse radish peroxidase (HRP). Incubations were for 1 h at room temperature. The plate is washed again a reacted with a solution of orthophenylenediamine (ODP), chromagen substrate for HRP. Absorbance in each well w measured at 490 nm and is reported in Table 3.
  • ODP orthophenylenediamine
  • Aqueous buffer is Autopow tissue culture media wit 8% horse serum, 2% fetal calf serum. All detergent used in the experiment were diluted in this buffer
  • Antibody No. 1 and Antibody 2 hav a relatively high affinity for Chlamydia in aqueous buffe that was not affected by any of the detergents except 2 NP-40.
  • Antibody 3 had a low affinity in 2% DOC, ye retained its high affinity in the other media.
  • Anti body 4 had a low affinity in 2% DOC and 2% NP-40 but high affinity in the other media.
  • the antigen coated microtite plates were first incubated with detergents in the concen trations shown in Table 3 for 1 hr. and then washed.
  • the antichlamydia antibodies were incubated i the wells followed, after washing, by an incubation with the HRP labeled anti-mouse antibodies. This incubation, after washing, was followed by an incubation with th enzyme substrate.
  • the optical densities measured in each well compared to wells which were pretreated with th aqueous buffer suggested that the antigen was not harmed by the detergents.
  • the monoclonal antibodies could be used for the affinity purification of the Chlamy ⁇ dia antigen by solubilizing the antigen in a detergen compatible with antibody binding and passing the prepara- tion over a column of immobilized antibody to bind the antigen. Subsequently, the antigen is released b eluting the column with another detergent composition i which the antibody does not bind to the antigen.
  • radiolabeled antibody used in an immunoradiometric assay which has degraded as a result of storage can be purified in this
  • the monoclonal antibody can also be recovered fro ascites fluid or culture medium by using the immobilize antigen as an immunoadsorbent.
  • the change in Ka with changes in pH is the likel effect of protonation of histidine residues or deprotona tion of lysine or possibly tyrosine or aginine residues i either the antibody, the antigen or both which alters th ability of the antigen and antibody to complex with eac other.
  • Specific residues affected may or may not li within the binding regions of the " molecules.
  • the antibodies produce by an animal's immune response to an antigen included antibodies that vary in their sensitivity to changes i environment, it is within the scope of our invention t fractionate polyclonal antisera to obtain a mixtur of antibodies which behave in a manner similar to th environmentally sensitive monoclonal antibodies of this invention.
  • This fractionation can be accomplished b contacting the immobilized antigen with an excess of th antisera in a first desired environmental condition followed by washing the immunoadsorbent to remove unbound material.
  • This step is followed by contacting the immno- adsorbent with a medium which is the second environment to elute antibodies which are not eluted under the first environmental condition.
  • the immobilized antigen is contacted with an excess of antisera at pH 7 and the immobilized antigen washed with a medium at pH 7 to remove antibodies which exhibit a low affinity at pH 7.
  • the immunoadsorbent is then eluted at pH 4 to remove anti ⁇ bodies which exhibit a low affinity at pH 4.
  • the eluant will contain the fraction of antibodies whose binding with the antigen is sensitive to changes in pH over the range pH 7 to pH 4. Similar fractionation can be done with urea and other inhibitors of antigen-antibody binding.
  • the resulting populations of antibodies may require further subfractionation to further remove those antibodies which elute due to an intrinsic low affinity rather than a Ka "switch". It is also within the scope of our invention to employ hybrid monoclonal antibodies having dual speci ⁇ ficities for affinity purification.
  • a process for ob ⁇ taining hybrid monoclonal* antibodies is described in the concurrently filed application of Martinis et al, "Anti- bodies Having Dual Specificities, Their Preparation And Uses Therefor", Serial No. —, (Lyon and Lyon Docket 162/98), the disclosure of which is incorporated by reference.
  • the hybrid monoclonal antibody has two specificities which may be for different antigens.
  • the hybrid antibody is selected to exhibit pH or other environmental sensitivity in the specificity for the antigen for which it is to be used as an immunoadsor ⁇ bent in an affinity chromatography.
  • the other specificity exhibited by the hybrid is selected to have a high affinity against a second antigen which is bound to a solid support.
  • the affinity of the hybrid for the second antigen must not be substantially lower in the environmental condition which will permit elution of the first, or target antigen.
  • the binding of second antigen to the antibody is sensitive to a different environmental condition than that which permits the target antigen to be eluted from the immuno ⁇ adsorbent.
  • the hybrid antibody may be selected so that the affinity for the target antigen is reduced by a lowering of pH and the affinity for the second antigen reduced by increasing the pH. This permits the hybrid monoclonal antibody to be desorbed readily from a solid support when it is desirable to do so because the support has become contaminated by impurities or oth reasons which impairs its usefulness.
  • the environmentally sensitive antibodies of ou invention can also be used to store antigens in a soli phase that are unstable in solution.
  • radio labeled antigen can be bound to the immobilized antibod for storage and desorbed as needed. Desorption can b preceded by washing the immunoadsorbent to remove an products of degration that arose during storage. Th reverse process is also possible, i.e., the antigen can b used to store unstable antibody in a solid phase.
  • radiolabeled antibody used in a radioassay can b stored as the antigen: antibody complex and desorbe as required.

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PCT/US1983/000524 1982-04-12 1983-04-12 Method of affinity purification employing monoclonal antibodies WO1983003678A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU15507/83A AU1550783A (en) 1982-04-12 1983-04-12 Method of affinity purification employing monoclonal antibodies
JP50166683A JPS59500720A (ja) 1983-04-12 1983-04-12 モノクロ−ナル抗体を用いるアフィニティ精製方法
GB08332644A GB2128630B (en) 1982-04-12 1983-04-12 Method of affinity purification employing monoclonal antibodies
DK564983A DK564983D0 (da) 1982-04-12 1983-12-08 Monoclonalt antistof og dets fremstilling og anvendelse i affinitets-rensningsprocesser
FI834530A FI88403C (fi) 1982-04-12 1983-12-09 Monoklonala antikroppar, foerfarande foer framstaellning av dessa och affinitetsreningsfoerfarande vari dessa anvaends
NO834535A NO168969C (no) 1982-04-12 1983-12-09 Fremgangsmaate for affinitetsrensing av et hybrid monoklonalt antistoff, fremgangsmaate for dets fremstilling og anvendelse av antistoffet
DK192191A DK192191A (da) 1982-04-12 1991-11-26 Monoclonalt antistof og dets fremstilling og anvendelse i affinitets-rensningsprocesser

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US36778182A 1982-04-12 1982-04-12
US367,781820412 1982-04-12

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AU (1) AU622099B2 (es)
CA (1) CA1209907A (es)
CH (1) CH672028A5 (es)
DK (1) DK192191A (es)
ES (3) ES8404858A1 (es)
FI (1) FI88403C (es)
GB (1) GB2128630B (es)
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WO (1) WO1983003678A1 (es)

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EP0145356A2 (en) * 1983-11-22 1985-06-19 National Research Development Corporation Method of assay of DNA or RNA from virus particles
WO1985003133A1 (en) * 1984-01-13 1985-07-18 Centocor, Inc. Immunoassay for biologically active human interferon-gamma
WO1986002355A1 (en) * 1984-10-19 1986-04-24 Technology Licence Company Limited Monoclonal antibodies and their use
EP0193431A1 (en) * 1985-01-30 1986-09-03 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Monoclonal antibodies against chlamydial genus specific lipopolysaccharide
US4659666A (en) * 1983-10-28 1987-04-21 Internationale Octrooi Maatschappij "Octropa" B.V. Pure alkaline phosphatase, its preparation and use
EP0290406A1 (en) * 1987-03-06 1988-11-09 Hyclone Laboratories, Inc. Low affinity adsorbent for affinity chromatography
US4789631A (en) * 1984-02-17 1988-12-06 Synbiotics Corporation Immunoassay for anti-dirofilaria immitis antibody
EP0293606A2 (en) * 1987-05-01 1988-12-07 Xoma Corporation Immunoglobulin purification by selective anti-idiotype binding
WO1989000695A1 (en) * 1987-07-14 1989-01-26 The Victoria University Of Manchester Method for the diagnosis of infections with detection of lipopolysaccharide antigens
US4818687A (en) * 1985-02-28 1989-04-04 Massachusetts Institute Of Technology Affinity column and process for detection of low molecular weight toxic substances
US4859611A (en) * 1985-02-28 1989-08-22 Massachusetts Institute Of Technology Affinity column and process for detection of low molecular weight toxic substances
US4876191A (en) * 1985-07-17 1989-10-24 Ramot University Authority For Applied Research And Industrial Development Ltd. Immobilization of biologically active substances with carrier bond antibody
US5175255A (en) * 1987-03-23 1992-12-29 Amgen Inc. Methods for purification of platelet-derived growth factor
US5190859A (en) * 1987-02-26 1993-03-02 Dana-Farber Cancer Institute, Inc. Purification of LFA-3
US5372812A (en) * 1988-04-04 1994-12-13 The General Hospital Corporation Composition and method for acceleration of clot lysis
US5552286A (en) * 1985-12-09 1996-09-03 Kirin-Amgen, Inc. Hybridoma cell lines and their monoclonal antibodies to human pluripotent granulocyte colony stimulating factor
US5582862A (en) * 1988-04-04 1996-12-10 General Hospital Corporation Antibodies that bind to α2-antiplasmin crosslinked to fibrin which do not inhibit plasma α2-antiplasmin
US6114506A (en) * 1996-09-20 2000-09-05 General Hospital Corporation Composition and method for enhancing fibrinolysis
CN110724204A (zh) * 2019-11-18 2020-01-24 深圳市菲鹏生物制药股份有限公司 Fc融合蛋白的纯化方法
US11359194B2 (en) * 2008-04-11 2022-06-14 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding two or more antigen molecules repeatedly
US11359009B2 (en) 2015-12-25 2022-06-14 Chugai Seiyaku Kabushiki Kaisha Anti-myostatin antibodies and methods of use
US11454633B2 (en) 2014-12-19 2022-09-27 Chugai Seiyaku Kabushiki Kaisha Anti-myostatin antibodies, polypeptides containing variant Fc regions, and methods of use
US11597760B2 (en) 2014-12-19 2023-03-07 Chugai Seiyaku Kabushiki Kaisha Method of detecting the presence of complement C5
US11608374B2 (en) 2017-01-30 2023-03-21 Chugai Seiyaku Kabushiki Kaisha Anti-sclerostin antibodies and methods of use
US11718678B2 (en) 2011-02-25 2023-08-08 Chugai Seiyaku Kabushiki Kaisha Method for altering plasma retention and immunogenicity of antigen-binding molecule
US11780912B2 (en) 2016-08-05 2023-10-10 Chugai Seiyaku Kabushiki Kaisha Composition for prophylaxis or treatment of IL-8 related diseases
US11820793B2 (en) 2011-11-30 2023-11-21 Chugai Seiyaku Kabushiki Kaisha Drug containing carrier into cell for forming immune complex
US11827699B2 (en) 2011-09-30 2023-11-28 Chugai Seiyaku Kabushiki Kaisha Methods for producing antibodies promoting disappearance of antigens having plurality of biological activities
US11891434B2 (en) 2010-11-30 2024-02-06 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly

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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4659666A (en) * 1983-10-28 1987-04-21 Internationale Octrooi Maatschappij "Octropa" B.V. Pure alkaline phosphatase, its preparation and use
EP0145356A2 (en) * 1983-11-22 1985-06-19 National Research Development Corporation Method of assay of DNA or RNA from virus particles
EP0145356A3 (en) * 1983-11-22 1985-07-17 National Research Development Corporation Method of extraction of dna or rna from virus particles and assay based thereon
WO1985003133A1 (en) * 1984-01-13 1985-07-18 Centocor, Inc. Immunoassay for biologically active human interferon-gamma
US4789631A (en) * 1984-02-17 1988-12-06 Synbiotics Corporation Immunoassay for anti-dirofilaria immitis antibody
WO1986002355A1 (en) * 1984-10-19 1986-04-24 Technology Licence Company Limited Monoclonal antibodies and their use
EP0193431A1 (en) * 1985-01-30 1986-09-03 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Monoclonal antibodies against chlamydial genus specific lipopolysaccharide
US4859611A (en) * 1985-02-28 1989-08-22 Massachusetts Institute Of Technology Affinity column and process for detection of low molecular weight toxic substances
US4818687A (en) * 1985-02-28 1989-04-04 Massachusetts Institute Of Technology Affinity column and process for detection of low molecular weight toxic substances
US4876191A (en) * 1985-07-17 1989-10-24 Ramot University Authority For Applied Research And Industrial Development Ltd. Immobilization of biologically active substances with carrier bond antibody
US5552286A (en) * 1985-12-09 1996-09-03 Kirin-Amgen, Inc. Hybridoma cell lines and their monoclonal antibodies to human pluripotent granulocyte colony stimulating factor
US5190859A (en) * 1987-02-26 1993-03-02 Dana-Farber Cancer Institute, Inc. Purification of LFA-3
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ES528000A0 (es) 1985-08-01
FI834530A (fi) 1983-12-09
EP0105359A1 (en) 1984-04-18
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ES528001A0 (es) 1987-07-01
FI88403C (fi) 1993-05-10
GB2128630A (en) 1984-05-02
DK192191D0 (da) 1991-11-26
IT1219779B (it) 1990-05-24
IT8320550A0 (it) 1983-04-12
GB2128630B (en) 1986-08-28
EP0105359A4 (en) 1984-08-20
GB8332644D0 (en) 1984-01-11
CA1209907A (en) 1986-08-19
ES8404858A1 (es) 1984-05-16
FI834530A0 (fi) 1983-12-09
ES8706964A1 (es) 1987-07-01
ES8506902A1 (es) 1985-08-01
ATA901983A (de) 1991-03-15
AU7256987A (en) 1987-09-03
DK192191A (da) 1991-11-26
AU622099B2 (en) 1992-04-02
AT393386B (de) 1991-10-10
FI88403B (fi) 1993-01-29

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