EP0494237A1 - Methode d'identification et de selection de cellules productrices d'anticorps - Google Patents

Methode d'identification et de selection de cellules productrices d'anticorps

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Publication number
EP0494237A1
EP0494237A1 EP19900915155 EP90915155A EP0494237A1 EP 0494237 A1 EP0494237 A1 EP 0494237A1 EP 19900915155 EP19900915155 EP 19900915155 EP 90915155 A EP90915155 A EP 90915155A EP 0494237 A1 EP0494237 A1 EP 0494237A1
Authority
EP
European Patent Office
Prior art keywords
antibody
cells
organisms
interest
filter layer
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19900915155
Other languages
German (de)
English (en)
Inventor
Cesar Milstein
Ermanno Gherardi
Martin Dreher
Richard Pannell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medical Research Council
Original Assignee
Medical Research Council
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 Medical Research Council filed Critical Medical Research Council
Publication of EP0494237A1 publication Critical patent/EP0494237A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran

Definitions

  • This invention concerns production of antibodies, and relates to a method of identifying antibody-producing organisms or cells which produce a particular antibody of interest.
  • hybridoma clones secreting antibodies of desired specificity is typically achieved in three steps: (1) the growth of hybridoma cultures, (2) the
  • screening methods must be capable of detecting a single positive clone out of perhaps 10 3 or 10 4 or more negative ones.
  • positive clones are mixed with negative ones, there is an increased probability of loss of valuable clones due to overgrowth by fast-growing negative competitors (Galfré et al.,
  • red cells or derivatised red cells in two ways: (1) Complement- dependent haemolytic plagues of red cells overlaid on developed clones (Kohler and Milstein, 1975) or mixed with hybridomas (Galfre and Milstein, 1981); (2) Binding of the red cells to a nitrocellulose replica containing adsorbed supernatant of the growing colonies (Sharon et al., 1979). Reference is also made to Kelsoe, 1987.
  • a method of identifying antibody-producing organisms or cells which produce a particular antibody of interest comprising locating individual organisms or cells on a surface in spaced-apart relationship; growing the
  • the antibody-producing organisms or cells may be any suitable antibody-producing organisms or cells.
  • prokaryolic organisms such as bacterial cells, and are preferably cells which secrete antibody either into the cell periplasmic space or from the cell completely into surrounding medium.
  • the organisms or cells may
  • eukaryotic cells eg mammalian cells such as mouse cells.
  • the antibody may be a complete antibody, including a bispecific antibody, or an antibody fragment such as an Fab or Fv fragment.
  • Suitable conditions for growth are selected depending on the nature of the organism or cell.
  • Eukaryotic cells are preferably grown in a feeder layer, which in some
  • embodiments conveniently comprises a layer of soft agarose containing STO fibroblast feeder cells.
  • the feeder layer is preferably covered with a diffusion layer, eg of agar: this assists clustering of cells derived from a particular individual initial cell during cell growth, and so helps maintain separation of different cell colonies.
  • a diffusion layer eg of agar: this assists clustering of cells derived from a particular individual initial cell during cell growth, and so helps maintain separation of different cell colonies.
  • the filter layer conveniently comprises nitrocellulose.
  • the reagent immobilized on the filter layer may be antigen to the antibody.
  • Antigen may be immobilized using known techniques, and may be bound to the layer directly or indirectly, eg via a secondary molecule such as another antibody, or using a biotin/avidin link.
  • the filter layer may be coated with anti-immunoglobulin or equivalent antibody capturing reagents.
  • Antibody of interest bound to immobilized reagent may be detected using known techniques, eg using radiolabelled or enzyme-labelled second antibody, as will be well known to those skilled in the art.
  • the organisms or cells After identification of organisms or cells producing the antibody of interest, by detection of bound antibodies, the organisms or cells may be selected and isolated.
  • Isolation may be achieved either by removing the organisms or cells from the surface, or by physically isolating the organisms or cells on the surface, eg by providing a suitable barrier separating the organisms or cells of interest from the surroundings. Isolated organisms or cells may be grown further or otherwise treated.
  • the method of the invention enables rapid screening and identification of organisms or cells producing antibody of interest, using a colony replica assay.
  • the method used can improve
  • antigen-specific hybridoma lines can be derived less than two weeks from the date of fusion.
  • the method of the invention can be used to discriminate on the basis of antigen affinity of antibodies, by screening with different amounts of antigen immobilized on the filter layer, eg using different filter layers each with different concentrations of antigen immobilized thereon.
  • the filter layers may be produced by linking antigen molecules to carrier molecules such as bovine serum albumin (BSA) or possibly another antibody in varying proportions (eg ranging from about 15 to about 1 molecules of antigen to each molecule of carrier) and coating the filter layer with a fixed constant amount of the antigen- carrier conjugate.
  • BSA bovine serum albumin
  • This technique provides a convenient means for achieving defined filter coatings carrying desired and variable amounts of antigen. This approach will generally be used to detect higher affinity
  • the present invention provides a method of selecting antibody-producing eukaryotic cells which produce a particular antibody of interest,
  • Another preferred aspect of the invention provides a method of identifying antibody-producing prokaryotic organisms which produce a particular antibody of interest, comprising locating individual organisms on a surface in spaced-apart relationship; growing the organisms under suitable conditions; applying to the organisms after growth a filter layer having immobilized thereon a reagent which binds to the antibody of interest; and detecting antibodies of interest bound to the filter layer.
  • Figure 1 is a replica assay of anti-hapten hybridoma clones cultured on STO fibroblast feeders in soft
  • Figure 2 is a replica assay of the anti-oxazolone
  • Figure 3 is a graph of 125 I-Rb anti-mouse 1gG bound (cpm x 10 3 /cm 2 ) versus Ox 14. 4 -BSA (pmoles/cm 2 );
  • Figure 4 is a replica assay of NQ2/16.2 hybridoma clones using an indirect assay with an anti-immunoglobulin coated filter layer;
  • Figure 5 is a comparison of results for direct and indirect replica assays.
  • Figure 6 illustrates schematically a bacterial colony assay for antibody fragments.
  • mice myeloma NSO (Clark and Milstein, 1981) and the mouse hybridoma NQ2/16.2 Ag.8 (an azaguanine-resistant clone of the hybridoma N02/16.2; Kaartinen et al., 1983) were cultured in DMEM + 5% FCS.
  • fibroblast line STO (Martin and Evans, 1975) was
  • STO feeders were produced by incubating subconfluent cultures in DMEM + 10% FCS supplemented with mytomicin C (Sigma M 0503, 10 ug/ml in DMEM + 10% FCS) for 2 hours at 37°C, after which cells were washed twice with PBS, lifted with trypsin-EDTA and melting point agarose (BRL 5517UB) in DMEM + 20% FCS.
  • Hybridomas were kept on ice for 10 mins before being returned to the incubator and were used, typically, 2 days after plating. Derivation of Hybridomas
  • Dishes were left on ice for 5-10 mins and returned to the incubator where they were left, undisturbed, for a week.
  • 8-16 cells were counted in at least 8 cm 2 across the plate.
  • nitrocellulose discs (Schleicher and Schuell, 40116) was performed overnight at 4oC using 1.3 nmoles of BSA/filter in 2.5 mis of PBS (this corresponds to different
  • asymmetric alignment marks were then made by piercing the nitrocellulose filter and the culture. The filters were then peeled off gently and transferred (face up) in 10 cm dishes containing 5 mis of PBS + 5% FCS + rabbit
  • Ox-BSA conjugates with 14.1, 3.1, 1.5 and 0.8 hapten/carrier molar ratios were prepared using different ratios of hapten/carrier (25, 10, 5 and 2.5 respectively) in the reaction.
  • KLH keyhole limpet haemocyanin
  • IgG monoclonal antibodies were purified from ascites or serum-free culture medium by affinity chromatography on Protein A-Sepharose 4B or Protein A-Superose columns
  • K d values were measured (using phenyl-oxazolone gamma-amino butirate) by fluorescence quenching, essentially as described by Eisen (1984). Results
  • Table 1 shows the results of a set of experiments in which mouse or rat spleen cells from animals which had received a primary immunisation with Ox-CSA or DNP-KLH were fused with the mouse myeloma NSO or the mouse hybridoma NQ2/16.2 Ag.8 and plated directly in soft agarose on STO feeders.
  • the number of growing clones varied considerably, as expected, from one experiment to another, but was always greater than 1,000 clones/10 8 spleen cells, and averaged 3554 clones/10 8 spleen cells in the set of experiments reported here. Each clone represents a different fusion event, since the cells were plated before expansion. This ensures maximum representation of the initial "library”.
  • FIG. 1 shows representative results of the procedure described, for fusions Q2R3 and NQS1. The figure shows the mirror image of antigen-specific clones from representative dishes.
  • the assay was performed with cultures less than 8 to 10 days old (established lines) or 10 to 12 days old (newly established hybridomas). When older cultures were used, a dark and uniform antibody staining appeared on the filter and the localised signal due to individual clones was lost.
  • Clones were classified as (A), to indicate a clone falling in the centre of the area marked, or (B), to indicate a peripheral clone. Sometimes two or more clones were too close to allow the identification of positive one(s). These were classified as (C), picked as a
  • Figure 2 shows an example of this approach.
  • Figure 2 is a replica assay of the anti- oxazolone hybridoma clones NQ2/16.2 and NQ11/7.12. Each cell line was subcloned in soft agarose (about 2500 cells/10 cm dish, without feeders) and assayed after 10 days using the colony replica assay. Nitrocellulose discs were coated with Ox 14. 4 -BSA (first lift), Ox 3 .1 -BSA
  • microculture is not only labour-intensive but also time- consuming. Clones must grow to a minimum cell density for supernatants to be assayed. During this period, the danger of negative variants arising with better growth characteristics is at its highest.
  • the ability to derive large numbers of independent clones after fusion is only useful if coupled to a rapid colony assay for the identification of the clones of interest.
  • the nitrocellulose is coated with the antigen of interest (Ox-BSA or DNP-BSA in our case), and blocked before being applied on the culture dishes.
  • This is suitable for antigens (such as proteins and nucleic acids) which can be immobilised on
  • nitrocellulose nitrocellulose.
  • Indirect methods are, however, more suitable for such cases and more generally applicable. This involves coating the nitrocellulose with anti-mouse (or rat) immunoglobulin and detection of antigen-specific clones by incubation with trace concentrations of labelled antigen. Direct adsorption of antibodies from the culture dish to uncoated nitrocellulose discs is very inefficient, probably due to competition with proteins in the culture medium (data not shown).
  • the replica assay has several other areas of application. For example, it would allow the rapid screening for bispecific antibodies, where the need for early cloning has been specifically emphasised (Suresh et al., 1986). Clones secreting bispecific antibodies could be detected by the binding of the antibodies to the first antigen (immobilised on nitrocellulose) followed by washing and incubation with a solution containing labelled second antigen. Another exciting application of the colony replica assay will be the screening of antigen-specific antibody fragments expressed in E. coli (Better et al., 1988; Skerra et al., 1988; Ward et al., 1989) or yeasts (Horwitz et al., 1988).
  • Antigen-binding Fab fragments secreted in the medium could be detected essentially with the procedure described.
  • the detection of Fv fragments will probably require several modifications of the basic protocol to ensure efficient detection of antigen-bound Fv.
  • the results of the direct colony replica assays described above for detection of anti-hapten antibodies were optimal when concentrations of hapten-carrier conjugate of 25 pmoles (carrier) per cm 2 were employed. With the hapten- carrier conjugate used in most experiments (Ox 14 .4 /BSA at a coupling ratio: 14.4/1) this corresponded to about 360 pmoles of hapten per cm 2 or about 20 nmoles of hapten per filter (8.2 cm diameter).
  • Figure 3 shows how some of the membranes performed in an assay which mimicked the direct colony assay. Squares of membranes were coated with different concentrations of Ox/BSA from 0 to 10 pmoles/cm 2 , blocked, incubated with the supernatant of an anti-hapten antibody (NQ2/16.2) and finally probed with 125 I- rabbit anti-mouse IgG, according to the following method.
  • Membranes were washed 3 x 5 mins with 0.25 ml/well of a solution of Tween 20 in phosphate buffered saline (1 g/1) and incubated for 1 hour with 125 I-rabbit anti-mouse IgG (50,000 pmole in 0.1 ml of 5% fetal calf serum in
  • Membranes were washed as carried out after the incubation with primary antibody, dried and counted.
  • NQ2/16.2 hybridoma cells were cloned in agarose in 10 cm dishes. Eight days after cloning, the dishes were
  • Membranes were blotted between two circles of Whatman filter paper n.1 and applied for 1 hour to the surface of culture dishes at 37°C before incubation for 1 hour with 1 x 10 6 cpm/ml of Ox/BSA.
  • Membranes were washed with lg/l Tween 20 in phosphate buffered saline (3 times x 5 mins), dried and exposed overnight at 70°C.
  • the PVDF membrane had the lowest background and the lowest signal but a good signal/background ratio.
  • the NQ2/16.2 hybridoma cells were cloned and used for colony replica assays as described in Gherardi et al.
  • Immobilon P membranes were coated for 8 - 10 hours at room temperature with either anti-immunoglobulin reagents or antigen, as described below, blocked for 1 hour at 37°C in 5% fetal calf serum in phosphate buffered saline, blotted between 2 circles of Whatman n.1 paper and applied for 1 hour on the surface of culture dishes which had been overlaid overnight with 2ml of 0.75% agar. Filters were then incubated for 1 hour with radiolabelled antigen or anti-immunoglobulin, washed three times x 5 mins with Tween 20 in phosphate buffered saline, dried and exposed overnight at 70oC.
  • the filters were coated and probed with the reagents listed in Table 4.
  • the concentration of probe was 2.5 x 10 6 cpm/ml in 2.5 ml/filter in each case.
  • bacteria are grown into colonies 10 on a nitrocellulose filter 12 placed on top of an agar plate 14 in a dish 16 with an appropriate antibiotic selection including 100 ug/ml Ampicillin in 1% glucose until a small colony has formed.
  • the nitrocellulose filter with bacterial colonies is transferred from the agar plate to another which has been supplemented with IPTG. This arrangement facilitates the handling of the bacteria in several ways:
  • All bacterial colonies are transferred in one step from the agar plate with the growth medium to another agar plate 18 with the components necessary for induction (ie production of the antibody fragments), including 100 ug/ml Ampicillin and 1 mM IPTG.
  • the filter can be used to produce an exact replica of the bacterial colonies. This allows duplicate screening to be performed.
  • Bacteria are partially immobilized on the filter matrix facilitating rescue of viable bacterial colonies in subsequent steps.
  • the filter 12 with the induced colonies 18 is covered with another filter 20 in the form of a 0.22 u membrane of low protein binding properties (eg Millipore, CatNo: GVWP 090 50) and overlaid with an antigen coated nitrocellulose filter 22.
  • the filters are placed on top of about 10 layers of 3MM Whatmann paper 24 soaked in osmotic shock buffer (0.2M NaBorate, 0.16M NaCl, pH8) and covered with about 20 layers of dry blotting paper 26.
  • the osmotic shock buffer diffuses through the filter with the
  • immobilized Fv-fragments on the antigen-coated filter can be visualized by, for instance, appropriate immunological reagents.
  • the technique offers the following advantages:
  • the desired antibody fragment can be found by screening such a
  • colony assays extends considerably the yield and recovery of antigen-specific clones. Similarly the colony assays described here have been used
  • antibodies (or antibody fragments)- captured on the membrane are incubated with mixtures of radiolabelled and unlabelled antigen in which the concentration of the unlabelled species is varied and set at values which allow binding of the trace label only to the high (or higher) affinity antibodies.
  • the choice of the capturing reagent in the indirect assay can be used to determine the isotype of the antibodies to be selected (for example after fusion or EBV
  • isotypes have to be selected from the beginning in view of particular applications.
  • Endothelial cell growth supplement A cell cloning factor that promotes the growth of monoclonal antibody producing hybridoma cells. J. Immunol. Methods 61, 195.

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Abstract

On identifie des organismes ou cellules (procaryotiques ou eucaryotiques) producteurs d'un anticorps précis en les plaçant séparément et à bonne distance les uns des autres sur une surface; on les cultive ensuite dans les conditions appropriées; puis on leur applique, une fois cultivés, un lit filtrant sur lequel est fixé un réactif qui s'unit à l'anticorps recherché; enfin, on repère les anticorps liés à l'antigène. On peut par la suite sélectionner et isoler, par exemple pour une nouvelle culture, les organismes ou cellules produisant l'anticorps recherché.
EP19900915155 1989-09-28 1990-09-26 Methode d'identification et de selection de cellules productrices d'anticorps Withdrawn EP0494237A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8921880 1989-09-28
GB898921880A GB8921880D0 (en) 1989-09-28 1989-09-28 Improvements in or relating to hybridomas

Publications (1)

Publication Number Publication Date
EP0494237A1 true EP0494237A1 (fr) 1992-07-15

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ID=10663738

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EP19900915155 Withdrawn EP0494237A1 (fr) 1989-09-28 1990-09-26 Methode d'identification et de selection de cellules productrices d'anticorps

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EP (1) EP0494237A1 (fr)
AU (1) AU6510990A (fr)
GB (1) GB8921880D0 (fr)
WO (1) WO1991005260A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2136092T3 (es) * 1991-09-23 1999-11-16 Medical Res Council Procedimientos para la produccion de anticuerpos humanizados.
FI93742C (fi) * 1992-10-23 1995-05-26 Elias Hakalehto Menetelmä ja laite solujen osoittamiseksi
AU687618B2 (en) * 1994-08-26 1998-02-26 University Of Sydney, The Detection of molecules associated with airborne particles
AUPM765894A0 (en) * 1994-08-26 1994-09-15 University Of Sydney, The Particle detection and identification
AUPP131098A0 (en) * 1998-01-13 1998-02-05 Bellon Pty Limited Identification of particles and macromolecular species
AUPR271901A0 (en) * 2001-01-25 2001-02-22 Inhalix Pty Ltd Collection and analysis of particles
IT1317108B1 (it) * 2000-12-06 2003-05-26 Philogen Srl Processo per la selezione di frammenti anticorporali anti-angiogenesi,frammenti anticorpali anti-angiogenesi cosi' ottenuti e loro uso.
GB0815675D0 (en) 2008-08-28 2008-10-08 Mabtech Ab Antibody secreting cell elispot

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4474893A (en) * 1981-07-01 1984-10-02 The University of Texas System Cancer Center Recombinant monoclonal antibodies
US4634676A (en) * 1984-06-06 1987-01-06 Becton, Dickinson And Company Replica plating device
AU4346989A (en) * 1988-10-14 1990-05-01 Cecil Czerkinsky A method for simultaneous detection of different types of antibodies and/or antigens produced by individual cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9105260A1 *

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Publication number Publication date
AU6510990A (en) 1991-04-28
WO1991005260A1 (fr) 1991-04-18
GB8921880D0 (en) 1989-11-15

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