Gene Therapy (1998) 5, 828–834
 1998 Stockton Press All rights reserved 0969-7128/98 $12.00
http://www.stockton-press.co.uk/gt
Systemic long-term delivery of antibodies in
immunocompetent animals using cellulose sulphate
capsules containing antibody-producing cells
¨
¨
M Pelegrin1, M Marin1, D Noel1, M Del Rio2, R Saller3, J Stange4, S Mitzner4, WH Gunzburg5 and
M Piechaczyk1
´ ´
´
Institut de Genetique Moleculaire de Montpellier, UMR 5535/IFR 24, Montpellier; 2UMR 9921, UFR Sciences Pharmaceutiques et
¨
¨
Biologiques, Montpellier, France; 3Bavarian Nordic, Munchen, Germany; 4Klinic fur Innere Medezin, Rostock, Germany; and
5
University of Veterinary Sciences, Vienna, Austria
1
Implantation of capsules containing antibody-producing
cells into patients would potentially permit systemic longterm delivery of antibodies and might, thus, be useful in
the development of surveillance treatments for cancers
and severe viral diseases. We show that cellulose sulphate
(CS) capsules containing hybridoma cells, when implanted
subcutaneously or in the intraperitoneal cavity, can be
used for delivering monoclonal antibodies into the bloodstream of immunocompetent mice for at least several
months. In contrast to capsules implanted into the intraperitoneal cavity, which remain mobile and nonvascularized,
capsules implanted under the skin form neo-organs which
become vascularized within days. This may explain the
higher blood concentration of the antibody we have
observed in the latter case. Importantly, neither an isolating
fibrosis nor an obvious inflammatory response was
detected at the capsule implantation sites during observation periods as long as 10 months. Finally, no antiidiotypic immune response against the ectopically delivered antibody was shown to occur. This rules out any
potent adjuvant effect of the cellulose sulphate matrix that
might have stimulated a neutralizing humoral response.
Taken together, our data indicate that encapsulation of
antibody-producing cells into CS might be used in antibody-based gene/cell therapy approaches.
Keywords: antibody delivery; anti-idiotypic response; capsules; hybridoma; cellulose sulphate matrix
Introduction
Provided that cytostatic or cytotoxic tumor-specific antibody genes are available, systemic delivery of antibodies
by engineered cells grafted to patients may be highly
valuable for long-term anti-cancer surveillance treatments to prevent relapse after a primary treatment such
as surgery, chemo- or radiotherapy. Such an approach
could also be used for treating severe viral diseases, such
as AIDS, if virus-neutralizing antibodies or antibodies
toxic for virus-producing cells are delivered. In addition,
long-term systemic delivery of antibodies may also be
useful for more fundamental purposes such as the development of new animal models of autoimmune diseases
in which the humoral response contributes to the development of the illness (see Ref. 1). Yet another possible
application could involve the development of a new cell
ablation technique useful for studying in vivo various differentiation pathways and/or the biological importance
of specific cell subsets. This could be achieved by the
release into the blood stream of cytotoxic antibodies recognizing cell type-specific membrane markers; in this situ´ ´
´
Correspondence: M Piechaczyk, Institut de Genetique Moleculaire de
Montpellier, UMR 5535/IFR 24, 1919 route de Mende, 34293 Montpellier
Cedex 05, France
Received 21 October 1997; accepted 18 December 1997
ation, antibodies would kill target cells, for instance after
a specific differentiation step, immediately following the
appearance of cognate antigens at the cell surface.
Using retroviral gene transfer, we have recently shown
that several cell types (including skin fibroblasts, myogenic cells and hepatocytes), amenable to genetic modification and grafting to patients, can produce antibodies
retaining the specificity and the affinity of the parental
antibody.2 Furthermore, the grafting of engineered myogenic cells allows the systemic delivery of cloned antibodies in the mouse for at least several months. Although
these observations lend support to the idea that engineering of patients’ cells may be useful for long-term antibody-based gene therapies,2 several issues potentially
limit the application of such a technology in the clinic.
First, such a therapeutic approach would be labour-intensive, time-consuming and costly given the present state
of the technology. Second, stable genetic modification of
patients’ cells currently utilizes ex vivo retroviral infection
followed by autologous grafting in order to avoid rejection of non-MHC-matched cells by the immune system.
This reduces the versatility of the approach since engineered cells from one individual cannot be used for
another. Third, efficient gene transfer and long-term
expression of transgenes in cells that can be used in gene
therapy protocols are issues that have not yet been completely solved.3–6
Antibody production by encapsulated cells
M Pelegrin et al
In this context, implantation into patients of engineered
cells encapsulated into immunoprotective devices7,8 may
represent a more versatile and cost-effective approach.
On the one hand, it should allow the same batch of nonMHC-matched cells (possibly selected in vitro for optimal
antibody expression) to be used for several patients and,
on the other, implantation of capsules is a simple surgical
operation. In addition, such a technique would also offer
the possibility of easy surgical removal of antibody-producing cells in case the treatment needed to be terminated. For optimal function, the capsule pores must meet
two criteria. First, they must be large enough to permit
both the exit of molecules of interest, such as antibodies,
and the entry and diffusion of nutrients necessary for cell
survival. Second, they must be small enough to prevent,
on one hand, the encapsulated cells from leaving the capsules and, on the other hand, cells of the host immune
system from entering the capsules. Several types of polymers, such as alginate,9 alginate–poly(l)lysine–alginate,8,10 various acrylic polymers11,12 and cellulose sulphate13 have already been used for encapsulating cells.
The various matrices differ in their physical and mechanical properties. Cellulose sulphate (CS) capsules seem to
offer several advantages over other capsule types. Particularly, their larger pore size allows the release of molecules as large as (or bigger than) antibodies.14 Moreover,
they show excellent mechanical properties, conferring
strength resistance important to withstand the mechanical stress of the implantation procedure as well as to
ensure long-lifetime in vivo. Finally, the encapsulation
technique is simple as compared with others,14 which is
an important consideration for large-scale production.
The aim of this study was to demonstrate the feasibility
of the systemic delivery of antibodies in living animals
by cells encapsulated in a CS matrix. Capsules containing
a hybridoma cell line (Tg10) secreting a mouse monoclonal antibody (mAb) directed against human thyroglobulin, used as a model system, were implanted subcutaneously and intraperitoneally. Reasons for the choice of
the Tg10 mAb included: (1) the availability of a sensitive
ELISA for monitoring its concentration in blood
samples;15,16 (2) the anti-idiotypic response which can be
induced against it has previously been studied;17 and (3)
the availability of the cDNAs encoding its heavy and
light chains would allow further genetic modification of
various cell types which can be used in gene/cell therapy
protocols2
involving capsule
implantation
(see
Discussion). Antibody production was detected for as
long as 4 months after subcutaneous implantation of the
encapsulated cells although this was accompanied by a
steady decrease with time correlating with hybridoma
cell death and not with capsule destruction, the latter
remaining intact for at least 10 months in vivo.
Importantly, no anti-idiotypic response against the Tg10
antibody was shown to occur. This rules out any potent
adjuvant effect of cellulose sulphate that may have led
to a humoral response against the Tg10 antibody. Taken
together, our data suggest that this method is suitable for
antibody-based gene therapy approaches directed against
cancers and viral diseases.
hybridoma cells or could affect antibody production.
Encapsulated cells, placed in cell culture conditions, were
counted at various time-points post-encapsulation after
crushing of capsules. Cell viability was assessed using
the trypan blue exclusion method. In parallel, Tg10 mAb
production was assayed by ELISA. The main observations were as follows: (1) each capsule, the diameter of
which varied from 0.5 to 2 mm with an average of 1 mm,
contained a mean of 2 × 104 Tg10 cells at the moment of
encapsulation; (2) the viability of encapsulated cells 4
days post-encapsulation was estimated to be at least 80%;
(3) production of the Tg10 mAb was detectable over a
period of 50 days (Figure 1); (4) an initial increase in antibody production lasting 10 days was observed and correlated with limited proliferation of Tg10 cells within capsules (which cannot exceed one or two rounds of division
because of space limitations) (Figure 1); and (5) a marked
drop in antibody production, which correlated with the
onset of cell death, began between day 10 and day 20
(Figure 1). Taken together, our data indicate that the
encapsulation process per se is not toxic for Tg10 cells.
Rather, cell survival within the CS matrix seems favoured
since Tg10 cells cultured in parallel under standard culture conditions but without passage died within 1–2
weeks.
Results
Figure 1 In vitro antibody production by CS-encapsulated Tg10
hybridoma cells. Twenty-five CS capsules containing Tg10 hybridoma
cells (mean of 2 × 104 cells per capsule) were cultured as described in
Materials and methods. The culture medium was replaced every 24 h, and
Tg10 mAb production was determined by ELISA on the days indicated.
Production of Tg10 is expressed in ng of antibody produced per capsule
per 24 h using the purified Tg10 mAb as a standard.
In vitro antibody production by Tg10 cells encapsulated
into cellulose sulphate
Our first aim was to determine whether the encapsulation process and the CS matrix could be toxic for Tg10
In vivo delivery of Tg10 mAb after implantation into
mice of Tg10 cell-containing CS capsules
We addressed whether systemic delivery of the Tg10
mAb could be achieved after implantation of capsules
into mice. Both subcutaneous (abdominal zone) and
intraperitoneal implantations were tested to determine
which localization would allow better delivery of antibodies into the bloodstream. Three C3H mice (mice 1, 2
and 3) were implanted intraperitoneally with six capsules
829
Antibody production by encapsulated cells
M Pelegrin et al
830
and one mouse (mouse 6) with 20 capsules (Figure 2a).
Similarly, three mice (mice 7, 8 and 9) were implanted
subcutaneously with six capsules and three mice (mice
10, 11 and 12) with 20 capsules (Figure 2b). Tg10 antibody
concentration in the bloodstream was subsequently
monitored by ELISA. Data presented in Figure 2 can be
summarized as follows: antibody production (1) is
directly correlated with the number of capsules
implanted, (2) reaches a peak between the second and
third week post-implantation, (3) is reproducibly higher
when capsules are implanted subcutaneously since it can
reach a value as high as 12.5 ␮g/ml as compared with
2.5 ␮g/ml in the case of intraperitoneal implantation, and
(4) can be detected for as long as 4 months although Tg10
antibody concentration in the blood progressively
dropped down to only several tens of ng/ml. Finally, the
decrease in the concentration of Tg10 antibody in the
serum seemed to be linked to progressive death of encapsulated cells and not just to antibody degradation after a
short period of production since, on the one hand, mouse
IgG1 half-life in vivo is approximately 4 days (Ref. 18 and
our unpublished data), which is a shorter time than that
required for disappearance of 50% of Tg10 in implanted
mice, and, on the other, viability of encapsulated cells in
capsules removed from mice 2 months after subcutaneous implantation was estimated to be 20–30%, which is
a value that correlates with the reduction in antibody
concentration.
Follow-up of the vascularization of capsules
Capsules implanted intraperitoneally and subcutaneously behave differently with regard to their vascularization. Thus, 2 months after implantation, subcutaneously
implanted capsules appear wrapped in a vascularized
pouch made up of loose connective tissue (Figure 3).
Importantly, formation of such neo-organs occurs regardless of whether capsules contain hybridoma cells or not.
In contrast, when capsules are implanted intraperitoneally, they do not form clusters but remain mobile and no
vascularization is observed. We next investigated the
onset of vascularization and whether the treatment of
capsules with angiogenic factors could accelerate and/or
improve the vascularization of subcutaneously implanted
capsules. Capsules treated with bFGF, a growth factor
long known to have angiogenic activity19,20 and/or type I
rat collagen, as described in Materials and methods, were
implanted and followed with respect to their vascularization. No obvious difference was observed between the
various experimental conditions tested. Blood vessels
developing around capsules were easily visible as early
as 3 days after implantation and complete vascularization
was reached after 2 to 3 weeks, with a network of blood
vessels being present both around and within the
neorgan remaining stable for at least 10 months (data
not shown).
No detectable inflammatory response developed at the
level of implanted Tg10 cell-containing CS capsules
Mice were examined for the development of possible
inflammatory responses triggered by implanted capsules.
No macroscopically detectable immediate or delayed
inflammatory response was observed in the vicinity of
capsules after intraperitoneal or subcutaneous implantation. Also noteworthy was the lack of change in the
vascularization of capsules even 10 months after subcutaneous implantation.
Figure 2 In vivo antibody production by CS-encapsulated Tg10
hybridoma cells. C3H mice were implanted with Tg10 cell-containing CS
capsules either intraperitoneally (a) or subcutaneously (b) as described in
Materials and methods. Six mice were implanted with six capsules (mice
1, 2, 3, 7, 8 and 9) and four mice with 20 capsules (mice 6, 10, 11 and
12). Tg10 mAb concentration in serum was assayed by ELISA on the
days indicated. Production of Tg10 mAb is expressed in ␮g/ml. Concentrations of Tg10 mAb in the blood of mice 10 and 12 at day 78 were 210
ng/ml and 320 ng/ml, respectively. Concentration of Tg10 mAb in mouse
11 at day 126 was 150 ng/ml.
Anti-idiotypic humoral response against the Tg10
antibody released by encapsulated cells was not
detectable
The possibility that a humoral response against the Tg10
antibody had developed in mice implanted with encapsulated hybridoma cells was also investigated. A previously characterized rabbit serum containing anti-Tg10
antibodies17 was used as a positive control for these
experiments. Additionally, a series of mice was immunized with the purified monoclonal antibody in the presence of Freund adjuvant, as described in Materials and
methods. Of the 13 mice injected, six developed a clear
anti-idiotypic response, indicating that mice are not
intrinsically refractory to induction of such a response
(Figure 4a). In contrast, no anti-idiotypic response was
Antibody production by encapsulated cells
M Pelegrin et al
831
Figure 3 Vascularization of capsules implanted subcutaneously (abdominal zone) at day 3, day 8, day 45 (× 20 magnitude) and day 60 (× 15 magnitude),
respectively, after implantation. Vascularization progressing to the inner part of the neoorgan is reproducibly observed as early as 15 days after
implantation.
detected in any of the mice implanted with capsules
regardless of the blood concentration of antibody and the
site of implantation (Figure 4b). These data indicate that
the CS matrix does not exert any obvious adjuvant effect
that would have favoured the induction of a neutralizing
humoral response against the Tg10 antibody.
Discussion
Using the Tg10 hybridoma cell line as a model system,
we show that long-term systemic delivery of antibodies
by cells encapsulated in a cellulose sulphate matrix is
possible. Antibody production in the blood was detectable for as long as 4 months after implantation of cellcontaining capsules with, however, a peak of production
between 2 and 3 weeks and a steady decrease thereafter.
The initial increase in antibody delivery is most likely
partially accounted for by cell growth within capsules,
the extent of which is limited because of space constraints, and the subsequent decrease correlates with progressive cell death. It seems likely that Tg10 cell death is
not a result of the encapsulation per se and/or of capsule
implantation but rather reflects poor survival capability
of the implanted cells. This poor capability can indeed be
observed both in vitro under standard cell culture conditions where the presence of macrophages is necessary
for reducing the fraction of cells undergoing apoptosis,
and in vivo, where the Tg10 cells poorly develop ascites in
immunodeficient mice. In contrast, the cellulose sulphate
polymer significantly extended Tg10 cell survival. An
interesting possibility for long-term delivery of antibody
would thus be the encapsulation of long-lived antibodyproducing cells rather than repeated implantation of cellcontaining capsules. Primary myoblasts, hepatocytes or
skin fibroblasts, which are all cell types capable of
secretion of antibodies upon genetic modification,2 are
candidates currently being tested for such a purpose.
Indeed, mouse fibroblasts have been shown to survive
for at least 1 year when implanted into mice in capsules
made up of alginate–poly-l-lysine–alginate.8
Polymers other than CS have also been used for encapsulating hybridoma cells. Rat hybridoma cells secreting
a mAb directed against murine IL-4 have been encapsulated in alginate and implanted intraperitoneally and
subcutaneously into mice.9 Serum antibody concentrations were comparable with those reached in our
experiments. However, the levels of antibody delivered
in the blood stream declined after 14 days as a consequence of capsule deterioration. Moreover, in this system
a 100% incidence of ascite development was observed 30
days after implantation as a result of cells released from
the capsules into the intraperitoneal cavity. Cellulose sulphate capsules thus offer an obvious advantage with
respect to mechanical resistance over alginate capsules
since they are malleable and can resist relatively high
pressure on them and despite careful analysis, we have
found no evidence that they can rupture in vivo. With
respect to the latter point, capsules were found intact as
long as 10 months after implantation regardless of
whether they were implanted subcutaneously or intraperitoneally and as long as 3 months after implantation
into the thymus, the pancreas and the mammary gland
(unpublished data). On the other hand, intraperitoneal
implantation into mice of hybridoma cells encapsulated
in an alginate–poly(l)lysine–alginate matrix has permitted delivery of anti-human IL6 antibodies at levels markedly higher21 than those we obtained for Tg10. One reason for this difference resides in the higher number of
cells that were implanted in Okada and coworkers’
experiments.21 Our data also support the notion that the
number of implanted cells is the primary limiting factor
for systemic antibody delivery (at least under the experimental conditions used) since Tg10 mAb serum concentrations roughly correlated with the number of capsules
used in the various experiments. Additionally, antibody
production by hybridoma cells was intrinsically better in
Antibody production by encapsulated cells
M Pelegrin et al
832
Figure 4 Anti-idiotypic response against the Tg10 mAb in immunized
mice and in mice implanted with encapsulated Tg10 cells. (a) Antiidiotypic response against the Tg10 mAb in mice immunized with purified
antibody. Experiments were conducted as described in Materials and
methods. Six of 13 mice immunized developed a marked anti-idiotypic
response (mice 1Im, 2Im, 3Im, 4Im, 5Im and 6Im). Two mice injected
with PBS were used as negative controls (mice 7Non-Im and 8Non-Im).
ELISA were performed using 1/250 dilutions of sera and values are
expressed in absorbance units. (b) Anti-idiotypic response against Tg10
mAb in mice implanted with Tg10 cell-containing CS capsules. Experiments were conducted as described in Materials and methods. No antiidiotypic response was detected either in the case of capsules implanted
intraperitoneally (mice 1, 2, 3 and 6) or subcutaneously (mice 7, 8, 9, 10,
11 and 12). ELISA were performed using 1/250 dilutions of sera and
values are expressed in absorbance units.
the case of Okada et al’s experiments.21 It must also be
taken into account that alginate–poly(l)lysine–alginate
capsules induce an inflammatory response22,23 and thus
it cannot be excluded that local release of cytokines by
activated macrophages could have stimulated antibody
production from the encapsulated cells. Interestingly, no
such inflammation is observed in the case of CS capsules.
Subcutaneous and intraperitoneal implantations of CS
cell-containing capsules revealed differences with respect
to at least two points. First, the amount of antibody
released in the bloodstream was markedly higher in the
former situation. A likely explanation for this difference
resides in the fact that capsules are rapidly vascularized
when implanted subcutaneously and are not vascularized at all when implanted intraperitoneally. In our
view, the beneficial effect of vascularization might be
two-fold: first, facilitating antibody uptake by blood; and
second, ensuring a better supply of nutrients favoring cell
survival since viability of cells within intraperitoneally
implanted capsules was reproducibly observed to be
lower. In addition to extensive vacularization, which
showed no significant alteration over the 10 months of
follow-up, the clustering of cells within a connective
pouch after subcutaneous implantation would allow
removal of capsules through an easy one-step surgical
ablation of the whole neo-organ should this prove necessary. Finally, it is important to underline that we have
never observed any isolating fibrosis developing around
implanted CS capsules even 10 months after implantation. This observation contrasts with that reported in
the case of alginate–poly(l)lysine–alginate microcapsules
around which a host reaction with fibrosis developed
(within 4 weeks), probably as a result of macrophage activation by the encapsulating polymer.22
Anti-idiotypic responses are often observed in patients
repeatedly treated with high doses of purified monoclonal antibodies and can sometimes, but not always,
neutralize the effects of the treatment.24 Moreover, the
mode of administration of antibodies has also been
shown to be a crucial parameter with regard to the induction of anti-idiotypic responses. For example, subcutaneous and intradermal injections have been reported to be
much more immunogenic than intravenous injection.25
Although the occurrence of an anti-idiotypic response
was unlikely in the case of the experiments conducted
by Savelkoul et al9 using alginate capsules, possible antiidiotypic responses occurring in mice implanted with
alginate–poly(l)lysine–alginate, hybridoma cell-containing capsules has unfortunately not been studied. It is
worth considering, however, that macrophages activated
in the vicinity of capsules could provide a milieu favoring the initiation of an anti-idiotypic response directed
against the antibody released by capsules. It is thus
important to underline that no detectable anti-idiotypic
response against the Tg10 mAb developed in mice
implanted with CS-encapsulated Tg10 cells. Since mice
are able to mount an anti-idiotypic response when properly immunized with the Tg10 mAb (ie in the presence
of Freund’s adjuvant), these data rule out any potent
adjuvant effect of the CS polymer that would favor the
initiation of an anti-antibody humoral response and
strengthen the idea that this polymer may be useful for
the development of long-term antibody-based gene/cell
therapy protocols.
Finally, the possibility of a cellular immune response
against encapsulated cells must also be taken into consideration. In the case of cell death, as we observed with
Tg10 hybridoma cells, debris released from capsules
could elicit an immune response. However, rather than
a drawback, we feel that such a response might reveal
an advantage with respect to safety. On the one hand,
production of antibodies by living encapsulated cells
should not be affected since T lymphocytes cannot enter
capsules and, on the other, encapsulated cells that would
incidentally be released from capsules would be killed
immediately. Experiments are currently underway to
exploit this possibility.
Antibody production by encapsulated cells
M Pelegrin et al
Materials and methods
Cells and cell culture conditions
The Tg10 hybridoma cell line has been described previously.15 Cells were grown in RPMI 1640 (GIBCO/BRL,
Cergy Pontoise, France) supplemented with 10% FCS in
the presence of 100 IU/ml streptomycin, 100 IU/ml of
penicillin and 2 mm l-glutamine. Tg10 cells encapsulated
in the CS matrix were cultured under the same conditions
as free cells.
Antibody and F(ab)⬘2 fragments purification
Tg10 mAb was purified for Tg10 cell culture supernatant
by affinity chromatography as previously described.26
Rabbit anti-idiotypic antibody against the Tg10 mAb was
produced as described in Del Rio et al.17 Tg10 F(ab)⬘2 fragment was prepared electrophoretically after cleavage of
Tg10 mAb by pepsin and purity of preparations was controlled by SDS-PAGE analysis.17
Cell encapsulation
Tg10 hybridoma cells were encapsulated as described
elsewhere.13 Briefly, 107 cells were resuspended in a 3.8%
sodium cellulose sulphate solution (kind gift from Dr
Dautzenberg, prepared as described in Ref. 14) and containing 5% fetal calf serum. The cell suspension was afterwards dropped into an agitated 2.5% poly-dimethyldiallyl-ammonium solution (PDDMDAAC; KatpolChemie, Bitterfield, Germany). Capsules, which formed
within 90 s were washed in DMEM.
Capsules implantation
Four- to six-week-old C3H mice (IFFA-CREDO) were
anaesthetized using 0.01 ml per gramme of body weight
of a solution containing 0.1% Xylazine (Rompun, Bayer,
`
ˆ
Germany) and 10 mg/ml Ketamine (Imalgene, Rhone
´
Merieux, Lyon, France). Capsules were washed in phosphate-buffered saline (0.15 m NaCl, 0.01 m Na phosphate
pH 7) before implantation. Six or twenty CSM-Tg10 capsules were implanted into mice either intraperitoneally
or subcutaneously (abdominal zone). In the latter case,
they were implanted at one or three sites in clusters of
six or seven capsules per site.
Mice immunization
For generating Tg10 anti-idiotypic antibodies, 6-week-old
B6D2F1 females (IFFA CREDO) were injected intradermally at multiple sites using 20 ␮g per mouse of purified Tg10 mAb emulsified in a 1:1 volume ratio of complete Freund’s adjuvant (Sigma). Two booster injections
of 20 ␮g of purified Tg10 mAb (10 ␮g intraperitoneally
and 10 ␮g intramuscularly) in incomplete Freund’s adjuvant (Sigma) were given 3 and 4 weeks after the first
immunization, respectively. Mice were bled before each
immunization and 1 week after the last one. After clotting, blood samples were centrifuged at 2900 g for 15 min
and serum aliquots were stored at −20°C until use. Each
serum sample was assayed for the presence of antiidiotypic antibody against Tg10 as described below.
Immunoassay of Tg10 antibodies: analysis of the antiidiotypic immune response
Tg10 antibodies in cell culture supernatants and in mouse
sera were assayed by ELISA using human thyroglobulin
(hTg) (UCB-Bioproducts, Belgium) for the coating of
microtiter plates (Maxisorb, Nunc, Strasbourg, France) as
¨
described in Piechaczyk et al15 and Noel et al16 and
absorbance at 450 nm was measured using the automated
MR5000 plate reader from Dynatech, France. Protein A
sepharose-purified Tg10 mAb was used as a standard for
concentration determination. Anti-idiotypic antibodies
against the Tg10 mAb were assayed by ELISA using Tg10
F(ab)⬘2 for the coating of microtiter plates as described in
Del Rio et al.17 A rabbit anti-idiotypic antiserum17 against
the Tg10 mAb was included in our experiments as a positive control.
Acknowledgements
This work was supported by grants from the Centre
National de la Recherche Scientifique, the Ligue Nationale contre le Cancer, the Association de Recherche contre
le Cancer (ARC), the Agence Nationale de Recherche contre le Sida (ANRS) and the EC Biotech Programme BIO
950100.
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