Article
Reference
Immunization in vitro and production of monoclonal antibodies specific to insoluble and weakly immunogenic proteins
VAN NESS, Jeffrey, LAEMMLI, Ulrich Karl, PETTIJOHN, David E.
Abstract
A procedure is described for immunizing in vitro and stimulating proliferation of specific B-cell lymphocytes. The method is applicable to production of monoclonal antibodies against proteins that are soluble only in denaturing solvents. An induction period is described in which antigen is presented to the B-cell population in the absence of serum. Also, antigen is coupled to mitogenic silica, which allows the effective presentation of both soluble and insoluble antigens. The results indicate hybridomas can be obtained that secrete IgMs directed against highly conserved or weakly immunogenic antigens.
VAN NESS, Jeffrey, LAEMMLI, Ulrich Karl, PETTIJOHN, David E. Immunization in vitro and production of monoclonal antibodies specific to insoluble and weakly immunogenic proteins.
Proceedings of the National Academy of Sciences, 1984, vol. 81, no. 24, p. 7897-7901
DOI : 10.1073/pnas.81.24.7897
Available at:
http://archive-ouverte.unige.ch/unige:127862
Disclaimer: layout of this document may differ from the published version.
Proc.Natl. Acad. Sci. USA
Vol. 81,pp.7897-7901, December 1984 Immunology
Immunization in vitro and production of monoclonal antibodies specific to insoluble and weakly immunogenic proteins
(hybridoma/mitogen/antigen presentation/chromosomal protein/immune suppression)
JEFFREY VAN NESS*, ULRICH K. LAEMMLI*, AND DAVID E. PETTUJOHNt
*University of Geneva,Departmentsof Molecular Biology andBiochemistry,20, QuaiErnest-Ansermet,Geneva,Switzerland;andtUniversity of Colorado Health SciencesCenter, Department ofBiochemistry/Biophysics/Genetics, 4200 East Ninth Avenue, Denver, CO 80262
Communicatedby David W. Talmage, August 6, 1984
ABSTRACT A procedure isdescribed for immunizing in vitro and stimulating proliferation of specific B-cell lympho- cytes. The method is applicable to production of monoclonal antibodiesagainst proteins that are soluble only in denaturing solvents. Aninduction period isdescribed in which antigen is presented to the B-cell population in the absence of serum.
Also, antigen is coupled tomitogenic silica, which allows the effectivepresentation of both soluble and insoluble antigens.
The resultsindicate hybridomas can be obtained that secrete IgMs directed against highlyconserved or weakly immunogen- ic antigens.
Nowthat thepotentialapplications of hybridoma technology areappreciated (for review, see ref. 1), the coupling ofin vitrostimulationof lymphocytes to hybridomaproduction is of greatimportance,asinvitroimmunization has several ad- vantages over in vivo methods. For example, (i) it ispossible to produceantibodies directed against highly conserved or weaklyimmunogenic determinants (2), (ii) comparatively lit- tleantigen is required (3), and (iii) in vitro stimulation (4, 5) will in manycases be the procedure required to generate hy- bridomas secreting human antibodies. Considerable ad- vances havebeen made recently in coupling the two proce- dures (2); however, several problems remain. The most sig- nificant is competition of introduced antigen with serum proteins or determinants in the rabbit or fetal calf serum re- quiredin mostsystems to support B-celldivision (6). Serum- free in vitrostimulationsystems areknown(7, 8), but these rely on replacements which themselves include other pro- teins orimmunogenic contaminants, and these systems are generally less supportive of B-cell division. Also, it has been difficult tointroduceinsolubleantigen into theinvitrostimu- lation reaction in a form that can be presented to the majority oflymphocytes or monocytes. Insoluble protein antigens ei- ther must besolubilized incytotoxic solvents, which must be avoidedduringin vitrostimulation, or must be used as pre- cipitates. Furthermore, the T-cell suppressor activity in the spleniccellpopulationideally should be eliminated, although it has been suggested that in vitro immunization is in part free of normal immunesuppression (6, 9). Here we describe studies directed at these problems.
METHODS
Coupling of Antigen to Silica. Metaphase chromosomes andnuclei were isolated from HeLa cells as described (10, 11) and associated nonhistone chromosomal proteins (NHCPs) were fractionated by NaDodSO4 gel electrophore- sis(10-12). Singlebands instainedpolyacrylamide gels (13) were cutfrom the gel with a razor blade. The protein was eluted from the gel slice essentially as described (14) by crushingthegelandmixing for24-48 hr with 1-5 mlof2%
NaDodSO4/1%2-mercaptoethanol/5 mMTris HCl, pH 7.2, and then was heated to 100'C for2 min. Thegel fragments were eliminated by centrifuging and filtering the solution through a Miller-HA filter (Millipore). Vimentin and a Mr 210,000 nucleolar protein weresimilarly preparedas single electrophoresisbands from HeLa cells. Total NHCPswere similarly solubilized in the above NaDodSO4 solution.
FumedSiO2 (10-1000
.g;
Sigma,size 0.007 ,um)suspended inwater by sonication was thenadded to the solubilized pro- tein and mixed for 12-24 hr at roomtemperature.Bindingto the beads was usually rapid, but in some cases, to insure better binding, solid CCl3COOH was added to afinalcon- centration of 25% (the latter step is required with certain proteins in NaDodSO4 solution). The silica beads with at- tachedprotein were collected bycentrifugation (10,000 x g for5min) and washedrepeatedly with distilledwater to re- moveNaDodSO4and/or CCl3COOH. More than90% of all '25I-labeledNHCP or vimentincosedimented with the silica.Prior to use as an immunogen, the silica-antigen complex was briefly sonicated and could be sterilized by irradiation orautoclaving.
Summaryof the in VitroStimulationReaction.Spleen cells, thymocytes(isolated byscreeningthroughwire mesh), and macrophages were isolated (the latterby peritoneal cavity wash) from four or fiveBALB/cmiceorC57/B1x BALB/c F1 hybrids (see refs. 16 and 17). Then, two sets of media were constructed. First, 5.0 ml of RPMI 1640mediumcon- taining 40ofetal calfserumand allofthe components de- scribed in Table 2 was thymocyte-conditioned by growing with 2 x 108autologousthymocytes per ml.Second,8ml of thesamemedium minus the fetal calfserumandthymocytes were used tosuspend the spleen cells (2.5 x 108), macro- phages (1 x 107),and 500
Mg
ofsilica-antigen complex.Both cellsuspensionswereplacedin25-cm2flasks(Coming)and incubated 7-9hr at 370C in 5% C02/95% air. The thymo- cyteswerethen removed from theserum-containingmedium bycentrifugation, and the mediumwasaddedtothespleen cellsuspension culture. Incubationcontinuedfor 5-8days.Theflaskwasnotdisturbedduringincubation.
Fusion withMyeloma Cells and Selection ofHybridomas.
Cells in the flaskweredecantedbygentleshaking. Both the myeloma (X63-Ag8.6.5.3, Ns-1, Sp2/0, or PAI-O) and spleen cell suspensions were washed twice at 370C with RPMI1640medium. The number ofnonreticulocyte splenic cellsexcludingtrypan blue wasdetermined, andtheywere mixed with an equal number ofmyelomacells and washed onemoretime. Thecellswerefused with37%polyethylene glycol (Merck,Mr4000)bydescribedprocedures (18).Cells were plated at aconcentrationof2-4 x 105 myelomas (de- terminedfromprefusion input)per microtiterplatewell(6- 12 x 105cells perml)in the presence of2 x 10 thymocytes per well in20%fetal calfserumwithout HAT components (hypoxanthine/thymidine/aminopterin). At 24 hr after the
Abbreviation: NHCP,nonhistone chromosomalprotein.
Thepublicationcostsof this articleweredefrayedin partbypagecharge payment.Thisarticle must therefore behereby marked "advertisement"
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7898 Immunology: Van Ness etaLP fusion,themedium wasreplaced with HAT medium(see ref.
17). Selection for HAT resistance was complete in 5-7 days dependingonthemyeloma. Screeningcommenced at 14-20 days afterfusion, asdescribed in Table 1. Selected colonies weresubclonedbylimiting dilution. We have noted no strik- ing differencesinfusion efficiency with any of the four my- elomas. Myelomas were routinely tested for mycoplasm contamination asthis is afrequentcauseof poor hybridoma efficiency.
RESULTS
Presentation ofAntigen. The most appropriate meansfor presentation ofantigen to lymphocytes in vitro are uncer- tain. The problemis exacerbated whenantigensare insolu- ble inphysiologicalbuffers. We chosetocoupleproteinanti- genstosmallparticulatesilica,assilica has been showntobe mitogenic (19-21) and possesses a property of binding stronglytoprotein. Proteinantigenswereboundtosilica in solvents in which the proteins are soluble topermit a uni- form distribution. The studies described here emphasize NHCPsasantigensand,inparticular,oneofthemajorpro- teins of the isolated chromosomal scaffoldcalled sc-i (10).
Bound protein was resistant to dissociation by repeated washinginphysiologicalbuffersorheatinginNaDodSO4/2- mercaptoethanol. Thesilica-proteinbeadswereintroduced into thein vitrostimulationreaction,wheretheydistributed uniformlyand effected theclumpingoflymphocytes.
Table 1. Theeffectofprimingin the absence of fetal calf serum onthenumber ofantigen-specificclones
Clones, Specific Antigen Exp. Induction no. clones,%
sc-i(2.5 Ag) 1 Yes(6Kr) 93 15
2 No 141 0
Vimentin (0.1jig) 1 Yes (9 hr) 97 71
2 No 117 47
Cu+2-NHCP(50jig) 1 Yes(8hr) 61 48
2 No 79 16
BALB/c x C57/B1F1mousespleencells(4 x 108)wereequally divided into two stimulationreactions,eachcontaining antigencou- pledtosilica. Onereactionmixture(Exp. 2,containing2x108 cells) was incubated continuously with thymocyte-conditioned medium containing fetal calf serum and all components described in Table2, theother reaction(Exp.1)undergoingthespecifiedinductionperiod (hoursspecifiedin the absenceof fetal calf serum)priortothe addi- tionofallogenic thymocyte-conditioned fetal calf serum. After 7-9 days ofgrowth, the lymphocytes were fused to PAI/Omyeloma cells, and hybridomas were grown in 96-well microtiter dishes.
Wellspositive for growth were screened for antibody secretionby filtering culture supernatants through nitrocellulose paperusinga filtration manifold (Schleicher&Schuell).The nitrocellulosesheet wasthenprobedforabsorbed mouse antibodiesbyincubatingwith 2 x 106 cpm of "2I-labeled sheep anti-mouse IgG (Fab fragment;
Amersham)asdescribed(11).The driednitrocellulose sheet was ex- posedtox-ray filmtoidentify by autoradiographydotscorrespond- ingtoantibody-producing hybridomas.Antibodiesdirected against specific proteinswerefurther screenedbyusingasimilar"dot-blot"
assay in which the purified protein immobilized on nitrocellulose was incubated in the manifold with hybridoma supernatants and washed, andthen the entire sheet was incubated with125I-labeled sheepanti-mouseIgG,allasjustdescribed.Hybridomas screening aspositivefor chromosomalproteinswerefurther screened for spe- cificbindingofantibodies-tonucleiorchromosomesbyanimmuno- fluorescence assay using fixed, permeabilized HeLa cells as de- scribed(11).Specificityof all of theanti-sc-1and someof the anti- vimentin antibodies wasconfirmed by usingtheimmunoblotassay (see Fig. 1). The number of specific clones described above are those secreting antibody (which average 20-30%o of total clones) binding onlythespecific protein(sc-i orvimentin)in theabove as- says. In the case ofCu+2-NHCP,specificitytonuclearantigenswas determinedonlywith theimmunofluorescence assay.
Theintroduction ofsilica alone stimulated lymphocytesto divide (ref. 21, Table 2). The basis for the mitogenicactivity of silica may be the indirect activation of macrophages as suggested (21), but this point remains unclear. Excessive free silica is cytotoxic.Themaximumamounttoleratedwas 500,ugper108 spleen cells, asalsopreviously found (21).
Assembly of Reaction Components. Itwasanticipatedthat themaximal response to a specific antigen might be obtained when the antigen was presented in theabsenceof themilieu of serum proteins. An induction or priming period was de- vised duringwhich antigen waspresented to thelymphocyte population in the absence of serum, but after whichthymo- cyte-conditioned medium containing serum was added to supportgrowth. Lymphocyteswereculturedin the absence of serum for up to 9 hr without affecting significantly the final number of responding and dividing cells, but the reper- toire ofresulting hybridomas was altered. Induction in the absence of fetal calf serum was required forproduction of significant numbers of clones specific for sc-i protein (Table 1). Infour separate attempts to produce sc-i-specific hybrid- omas, none were obtained when immunization was in the presence offetal calf serum without the induction period, andmany wereobtained when the induction in the absence of fetal calf serum was used. However, the possibility that the induction periodmight be lessimportant at high concen- trations of antigen has not been investigated. Although the numberof clonesspecific for vimentin- or copper-stabilized NHCP was increased by the use of theinductionperiod,sub- stantial numbers of clones specific to theantigens alsowere observed without the fetal calf serum-free induction period.
Since sc-i protein is a relatively weak immunogen and the othertested proteins are stronger (seebelow), studiestosee if the apparent correlation between the valueofserum-free priming and immunogenicity of the antigen is observed should be carried out.
Components of the Reaction. Table 2 summarizes the rela- tiveeffects ofgrowth-supporting factors and antisuppressor T-cell drugs. It has been shown that suppressor T cells, which arenormal components of the spleen cell population, are capable of inhibiting specific antibody production in vi- tro (22). Inhibition of suppressorT-cell activity would be of obvious value, especially when attemptingtogenerateare- sponse tohighly conservedprotein. Cimetidine acts onsup- pressor T cells bearing the surface receptordesignatedH2, which are histamine binding (23, 24) and may abrogate the suppressoreffect (27). 1,1-Dimethylhydrazineandhydrocor- tisone are used to generate awiderspectrumofantisuppres- sor activity, while at low concentrationsleaving B-cellsun- affected (28, 29). Glutathione and 2-mercaptoethanol are added asgrowth factors (21, 30). Nonessential aminoacids, glutamine, and pyruvate arecommonly usedas lymphocyte supporting factors (6). In different experiments, the added nucleosides increased theyield of stimulated cells by2- to 6- fold. The addition ofthymocyte-conditioned medium after the priming period was absolutely required to support lym- phocytedivision. Cell concentrationwasimportant. Optimal response occurred at spleencell concentrations of1 x 108to 6 x 108 cells per 25 cm2. At lower cell concentrations, the number of stimulated cells fell quickly to 10% of maximal (data not shown).
Directing the Lymphocyte Response. Three specific anti- gens werestudied with respectto(i) theirabilitytostimulate lymphocytes, (ii) theirrespective required concentration to elicit response, and(iii) theeffects ofthepurity of the intro- duced antigen on specific response (Table 3). The antigens differed greatly in their ability to stimulate lymphocytes.
There was, for example, anapparentdifference of104inmo- lar concentration ofantigenrequired toobtain areasonable probabilityofobtainingananti-sc-1 clonecomparedwith ob- tainingananti-vimentin clone. When the datawere normal-
Proc. NatL Acad Sci. USA 81
(1984)
Proc. Natl. Acad. Sci. USA 81 (1984) 7899 Table 2. Effect of different components of the in vitro medium on the number of stimulated lymphocytes
Cells per Stimulated cells
Exp. Conditions Colonies, no. colony, no. Total no. Relative no.
A Fetal calf serum 3.2 x 102 3-4 1.3 x 103 1
B Thymocyte-conditioned
fetal calf serum 1.4 x 8 1.1 x 8.4
C Exp. B
+ cimetidine 8.6 x 103 7 6.0x 1i4 46
D Exp.C
+hydrocortisone
+ dimethylhydrazine 7.2 x 103 6 4.3 x 104 33
E Exp. D
+ mercaptoethanol
+glutathione 14.1 x 103 9 1.3 x 105 100
F Exp. E
+ nucleosides
+ hypoxanthine 28.1 x 103 8 2.2 x 105 169
G Exp. C
+ nucleosides 12.3 x 103 6 7.4 x 104 56
BALB/cx C57/B1 F1mouse spleen cells (3 x 106)wereincubated in 0.2 ml of RPMI 1640 medium containing 100 units per ml ofpenicillin/
streptomycin, 2 mM sodium pyruvate, 1 mM sodium glutamine, and nonessential amino acids solution at1x (GIBCO;supplied at100x) in the presenceof 1AgofCu'2-fixed NHCP attached to 0.1MgofSiO2. Othersimilar mixtures had additions asdescribed above at the following concentrations: 100AMcimetidine (Sigma); 1MMhydrocortisone (Sigma);1,1-dimethylhydrazine(assymetric form, Fluka) at 10Mg/ml;20,uM 2-mercaptoethanol(Sigma); 20AMglutathione (Sigma); 20MMeach adenosine, guanosine, uridine, and cytidine;3
AM
thymidine; and 30AM
hypoxanthine. The plates were incubated 5 days, and the number of plaque-forming colonies weredetermined;numbers being averaged from 12 identical wells in two separateexperiments.The total number ofstimulated cells was determined from the average number of cells per colony (3-4 cells per colonyorgreater, 30determinations per condition) multiplied by the number of plaque-forming colonies. The numbers presented may represent anunderestimate, as single or double "stimulated cells" would go unscored. An "induction"period of 8 hr was used asdescribed intextpriortothe additionof thymocyte-conditioned medium. At5days ofincubation, the stimulated cells were easily discerned as colonies or clustersof putative daughter cells.
ized on a per hybridoma basis, vimentin was 5-10 x 104 times moreimmunogenic permol than was sc-i. The nucleo- lar antigenrepresents an immunogenic value between the ex- tremesof sc-i and vimentin. Thereappears tobeanapproxi- mate correlation between eliciting amounts ofantigen and thequantitiesof stimulated cells and, hence, specific hybrid- omas.
Whencomplex mixtures ofantigensarepresentedtolym- phocytesinvitro,response tocertainantigensis reduced.To examinethis,Cu2+-fixed NHCPsweretested,asthisprotein population consists essentially of all chromosomal proteins (58 as detected by electrophoresis) devoid of histones and proteinsunabletobecrosslinkedby Cu2+(10). Althoughsc- 1proteinrepresents oneof themajor proteincomponents of Cu+2-fixed NHCPs(10), the abilityof sc-i to act as an im- munogen wasreducedinthepresenceof otherproteins. For example, one can compare the number ofsc-l-specific hy- bridomasproducedwhenpurifiedsc-iwasusedasimmuno- gen orwhen anequivalentamount ofsc-i mixed with other NHCPs was used instead. Since sc-1 comprised5-10% of the total NHCPs, it seemsthat the immunogenicityofsc-i was masked by or in competition with the other proteins.
Therefore,purified antigenwasused whenpossible. Stimu- latingspleen cells withcomplexmixturesof NHCPs resulted incomplexpatternsofhybridoma activities,with the excep- tion ofa preponderance ofantibodies directed against the nucleolus (30-35% of differentactivities) anddiverse cyto- plasmic antigens(40-45%) (data notshown).
Characterization ofProducingHybridomasandAntibodies.
The in vitro stimulation oflymphocytesinduced only apri- mary response;hence, >99% of all thehybridomasobtained byin vitrostimulationprotocols secreted IgM(Table 3).We produced IgG-secreting hybridomas but at present cannot rule out the possibility that a preexisting activity in the mousespleen cellpopulationwasimmortalized. IgM-secret- ing hybridomas are stable; twice-cloned hybridomas have been culturedfor6monthscontinuouslywithout diminution ofactivity. Monoclonalantibodiesproducedbythis proce-
durewerespecificfor respective immunogens by the differ- entassays described in Table 1. For example, an immuno- blot analysis of total human chromosomal proteins using a typicalanti-sc-iantibodyshowedaclear reaction withapro- teinhaving theelectrophoreticmobilityofsc-ibutnodetect- able reaction with any other protein (Fig. 1). Most of the antibodiesrecognizingotherantigens had similarspecificity to asingle protein. The antibodies generallywereapplicable tomanyexperimentsrequiringprecise recognition ofaspe- cific protein. Forexample, antibodiestaggedwithcolloidal goldorwithfluorescencewereusedtolocalize the intranu- clear and intrachromosomal positions of sc-1 protein via electronand fluorescencemicroscopy.Thesestudies will be described elsewhere.
Antibodies directedagainstsc-1, theMr210,000 nucleolar antigen, or vimentin reacted with antigens present in all mammalian cell lines tested in the immunofluorescence as- say. These cell lines included human (HeLa53; CCL2.2), Chinesehamsterovarycell(CHO-Ki;CCL2.2), andmouse
(X63-AG8.6.5.3). Forexample,18of 19 differentanti-nucle- olar andanti-nuclear-chromosomalantigen-specificantibod- ies reactedacross aspectrumof mammalian species, asdid nine of nine antibodies specific for cytoplasmic antigens.
When theseresultswerecompared with the activities of hy- bridomasproducedfromin vivoimmunization with identical antigens, a notable difference was seen. On average, only 50% ofanti-nuclear/nucleolaractivities reacted acrossspe- cies lines, andanti-chromosomal activities wererarely ob- tained(1%ofproducing cloneswhenNHCPswere used as antigen; unpublished data). Similar results afterin vivoim- munization have been described (32). Thus, it would seem
that antibodies produced bythe in vitro andin vivo proce- dure areprimarily directed against different classes ofepi-
topes innuclearproteins.
DISCUSSION
We have describedproceduresfor stimulating lymphocytes in vitrowithweaklyimmunogenic,insolubleantigensandfor
Immunology:
VanNess etaL7900 Immunology: Van Ness et al.
Table 3. Dependence of the numbers of specific hybridomasonthe concentration ofantigen Quantity Stimulated Clonessecreting Positive Specific
of Clones,* cells,t
antibodyj
clones,§clones,$
antigen,pg no. no. no. S % Subtype
sc-i0.05 43 1.2 x 106 10 60 0 IgM
0.1 68 4.6 x 106 17 76 0 IgM
1.0 59 6.8 x 106 12 75 0 IgM
2.5 93 5.1 x 106 18 67 15 IgM
5 87 6.9 x 106 21 76 12 IgM
M,210,000 nucleolar
0.001 115 3.5 x 106 21 71 6 1gM
0.05 78 4.5 x 106 15 67 50 IgM
0.1 161 4.1 x 106 28 71 39 IgM
1 86 6.7 x 106 16 56 55 IgM
Vimentin
0.001 79 4.6 x 106 23 65 67 IgM
0.1 98 8.6 x 106 17 53 77 IgM
1 119 1.07 x 107 31 81 64 IgM +IgG
Silica
1 15 1.0 x 106 7 0 0 NT
10 26 0.9 x 106 5 0 0 NT
100 18 1.3 x 106 5 0 0 NT
500 6 0.8 x 106 0 0 0 NT
Cu+2-NHCP
1 67 5 x 106 17 65 0 IgM
10 88 5.8 x106 19 47 0 IgM
50 61 4.0 x 106 23 69 0 IgM
100 72 8 x106 28 54 6 IgM
Spleencellsfrom sixBALB/cx c57/B1F1mice(5-6x 108)werepreparedandstimulated 8 hr in the presenceof thequantitiesof antigen listed above coupled to 500 ,ug of silica. Media contained all of theadditives described in Table 2. After growing 8 days, spleen cells were fused withPAI/Omyeloma cells. Ig classwasdeterminedbyOuchterlonydiffusion (31). NT,nottested.
*Numberof estimated clones per fusionsurvivingpastthe16-cellcolony size.
tNumberof nonreticulocyte spleen cells surviving5-6days in culture and excluding trypan blue.
tNumberof total clonessecretingantibody.
§Percentageoforiginalclonessecreting antibodyreacting in the dot-blot assayand/orimmunofluores- cenceassayand, therefore, secreting antibodyreacting withsomeprotein derivedfrom HeLa cells.
VPercentage of positive clones secretingantibodythat react via immunoblot assay specifically with respective antigen. In thecaseofCu+2-NHCP, the specific clones are those producing antibodies specificfor sc-1.
the
production
ofspecific
monoclonal antibodies. Theproto- col can be discussed in terms of five critical parameters.First,
aninductionperiod
is usedtodefinetherepertoire
of response of thecapable lymphocytes.
The in vitropriming
occursintheabsence ofserum and, therefore, reducesthe
possible problems
ofcompetition by
serumcomponents. Itseemsthat
only
themosthighly immunogenic antigens
elicita
significant
response in the presence of fetal calfserum.Second,
theuseofantigen coupled
tofumed silica apparent-ly
allowsappropriate presentation
ofantigen. Both soluble andinsolubleantigens
arereadily
andirreversiblyboundtosilica,
and theresulting
complex iseasily dispersedinto the stimulation reaction. Theantigen-silica
complexinduces ag-gregation
oflymphocytes
and, thus, enhances theprobablerequirement
of direct cell-cell contactand antigen binding(33, 34).
Silica itselfmay actas amacrophageattractantoractivator,
thustriggering
theputative requirement
formac-rophage
involvement in Tcell-mediated responses(35-38).Third,
we have noted arequirement
for seemingly highcell concentrationsduring lymphocyte
stimulation (39), but wedonot noteanarrowrangeof cell concentrations critical for response. It seems
probable
that the initial silica-inducedclustering
of cells isresponsible
for this lesscriticalrequire-
ment. Fourth, acomplex setofanti-suppressor T-cell drugs andnonspecific growthfactors isincludedduringthe stimu- lationperiod. Wehavenotestablished whether the anti-sup- pressor drugs significantly expand the repertoire of the preimmune spleen,buttheconcept appearsworthy of study.
Anabsoluterequirementforthymocyte-conditioned medium isshown,butautologousaswell asheterologousthymocytes arecapableofconditioningmedia and wehave not adopted the use ofmixed-thymocyte conditioning. Inthisprocedure there exists no particular requirement for theuseof prese- lected fetal calfserum for supporting lymphocyte division, which isprobablyduetotheuseofglutathione (40). Rabbit serumhas been reportedas asuccessful substitute for fetal calfserum. We have foundallogenicandautologous serato beinhibitory,which is inagreementwithotherfindings (41).
Fifth, we have critically reexamined the current paradigm that extraordinarily smallquantitiesofantigenare required toelicitaresponsein vitro(3).The datapresented appearsto indicate that, although in some cases nanogram levels of antigenare immunogenic, different protein antigens, espe- cially if conserved through evolution, require substantially higher quantities.
The response to antigen elicitedin vitro is a primary re- Proc. NatL Acad Sci. USA81
(1984)
Proc. NatL Acad. Sci USA 81 (1984) 7901
205-
116- 97-
Ln Ln
E E
o o
0 0
a E s E '-
ah._u--Sc
-C0-'
2:U 0z-m
~a a
-29---
FIG. 1. Immunoblot analysis ofanti-sc-1 antibodies. HeLameta-
phase chromosomesandinterphase nucleiwereprepared, andpro- teinswereisolated. Identicalamountsofproteinsweresubjectedto electrophoresis in gradient 7.5-15% polyacrylamide gels. (Right) Halfof thegelwastransferredtonitrocellulose andimmunoprobed with ananti-sc-1 antibody and '251-labeled secondary antibodyas described(11). (Left)Theotherhalf of thegel containingthesame
proteinswasstained forvisualization oftotalproteins.Thepositions ofmarkerproteins (in kDa)and of thesc-i proteinof170 kDaare
indicated.
sponse;hence, wehave immortalized primarilyactivitiesin theIgM subclass. It has been suggested that IgG-secreting hybridomascanbeobtainedbyextendingthe incubationpe-
riodto6or7daysandrestimulatingthelymphocyteculture (2). We have beenlargelyunsuccessfulatattemptstoobtain IgG-secreting hybridomas byextended culture but otherpro-
tocolscan be suggested.
Recently, preliminary results with this procedure incol- laborationwith PeterBrams and Karen Linnetinthe labora- toryofLennart Olsson atthe State University Hospital in Copenhagenhave indicated thathighly specific monoclonal antibodieswere obtainedagainstamammaliancorehistone and a subunitof the insulin receptor. Thus, it appearsthis procedureisapplicabletoother insolubleand weak immuno-
gens derivedfrom both chromosomes andmembranes.
We thank Professors Robert Lasher and LennartOls$onforin- valuablecommentsandsuggestionsand MartinTurman forhelpful discussions. The workwassupported bygrantstoD.E.P.from U.S.
National Institutes of Health(GM18243-12)and from the U.S. Na- tional ScienceFoundation (PCM-7921406)andto U.K.L.from the Swiss National ScienceFoundation(3.239.82).
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