From the Department of Medical Microbiology,
University Medical Center St. Radboud, 6500 HB
Nijmegen, The Netherlands, ¶ Department of Biochemistry,
University of Nijmegen, 6500 HB Nijmegen, The Netherlands, and ** Dyax
B.V., 6202 AZ Maastricht, The Netherlands
Received for publication, January 22, 2001, and in revised form, February 12, 2001
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ABSTRACT |
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We report the first construction of two combinatorial
human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete
surface protein of the sexual stages of Plasmodium
falciparum, were selected and analyzed extensively. The selected
scFv reacted with the surface of extracellular sexual forms of the
parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from
gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45
exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one VH family-derived
germ-line gene (VH1) and two VL family segments
(VL2 and VKI).
Plasmodium malaria is transmitted to the
Anopheles vector when mosquitoes ingest blood that contains
gametocytes. Gametocytes of Plasmodium falciparum synthesize
Pfs230 and Pfs48/45, which are expressed on the surface of macrogametes
and zygotes and a target for transmission-blocking immunity.
Antibodies in the ingested bloodmeal can bind to sexual forms in the
mosquito gut and prevent oocyst development (1-3). A panel of murine
and rat monoclonal antibodies
(mAbs)1 has been produced against
Pfs48/45 and has recognized at least five different epitopes. Some of
these mAbs showed transmission-blocking activity (3-6).
Transmission-blocking vaccines directed against sexual stage-specific
antigens are designed to arrest the development of sporogonic stages
inside the mosquito, thereby reducing the infectivity of the mosquito
and thereby prohibiting the spread of the disease. Major
obstacles in developing a transmission-blocking vaccine are the
production of correctly folded Pfs48/45 because of the conformational
nature of the Pfs48/45 epitope. A large panel of anti-Pfs48/45 mAbs was
needed for a number of reasons: 1) to elucidate the relationship
between Pfs48/45 and Pfs230 and 2) to measure and characterize
anti-Pfs48/45 antibodies in experimental and field sera.
Phage display antibodies offer a method for the production of high
affinity single chain variable fragment (scFv) derivatives of
human antibodies of "natural host" origin (for reviews, see Ref.
7). Our objective was to produce human mAbs against Pfs48/45, a sexual
stage antigen of P. falciparum. For this study combinatorial phage display libraries were constructed using B-lymphocytes from P. falciparum gametocyte carriers with transmission-blocking
immunity. Subsequently, phages were selected from these libraries by
panning on Pfs48/45 antigen and phages bound to the antigen
eluted by competition with mAbs (8). This method resulted in
human scFv antibodies directed against epitope III of Pfs48/45.
Parasites--
Mature P. falciparum gametocytes (NF54
strain) were produced in a semi-automated system as described
previously (12). Gametocytes were isolated as described previously
(13). The purified gametocytes were (a) used directly, in
immunofluorescence assays (see below), or (b) stored at
Gametocyte antigens were extracted using 25 mM Tris-HCl (pH
8.0) supplemented with 150 mM NaCl, 1.0% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each DNase
and RNase. Insoluble debris was pelleted by centrifugation (16,000 × g for 5 min at room temperature), and the supernatant was
stored at Monoclonal Antibodies--
Anti-Pfs48/45 murine mAbs 32F3
(IgG1; epitope Ia) and 32F1 (IgG2a; epitope
IIb) and rat mAbs 85RF45.1, 85RF45.2b, 85RF45.3, and 85RF45.5
(recognizing epitope I, IIb, III, and V of Pfs48/45) have been
described previously (3, 6).
mAb P5D4 recognizes the C-terminal vesicular stomatitis virus
glycoprotein (VSV-G)(14). Labeling of mAb P5D4 with horseradish peroxidase (HRP) was performed using the periodate method with a
molar input HRP/IgG ratio of 4 (15). The labeled mAb was dialyzed against PBS, supplemented with thimerosal (0.01%) and fetal calf serum (1%), and stored at Phage Libraries and Selection Procedures--
In this study, two
human phage display combinatorial immune antibody libraries were used.
The scFv library was derived from RNA obtained from lymphocytes of
(a) 10 gametocyte carriers from Cameroon after pooling of
the lymphocytes (Cam; 80 × 106 cells); and
(b) a Dutch expatriate (Spa; 45 × 106
cells) living in Cameroon for more than 30 years with regular attacks
of clinical malaria. The scFv library was made essentially as described
by Marks et al. (16) and Hoet et al. (9).
We used a two-step cloning procedure whereby the heavy and light chain
repertoires were cloned sequentially in the phagemid vector pHENIX
containing the VSV-G tag (14).2
Library diversity was analyzed by PCR and fingerprinting
(BstNI digestion).
The phage libraries were panned for binders using immunotubes (Nunc,
Maxisorp) coated with a Nonidet P-40 extract of gametocytes (500,000 parasite equivalents/tube). Elution of antigen binding clones was
performed by competition with a mixture of four rat mAbs (concentration
of 60 µg/ml each) (recognizing epitope I, IIb, III, and V of
Pfs48/45) for 90 min. The eluted phages were then allowed to infect
Escherichia coli TG1 host cells to amplify selected phage
binding to Pfs48/45. After amplification phages were selected for two
additional rounds using the same protocol. An aliquot of each of the
polyclonal phages obtained after each round of selection was stored at
4 °C until required.
After each round of selection, 96 single clones were screened for
binding to Pfs48/45 by ELISA. Clones of interest were characterized by:
(a) PCR-fingerprinting using the restriction enzyme
BstNI, (b) competition ELISA, (c)
sequencing, (d) immunofluorescence, and (e)
Western immunoblot (see below). Periplasmatic soluble scFv antibodies
and phage antibodies of different clones were produced as described by
Marks et al. (16).
ELISA Screening--
Pfs48/45-specific ELISA was performed using
a two-site ELISA as described previously (10). Briefly, microtiter
plates were coated with 50 µl of anti-Pfs48/45 mouse mAbs recognizing
different epitopes (10 µg/ml) in PBS for 30 min. After washing and
incubation with parasite extract (200,000 parasite equivalents/well),
50 µl of bacterial culture supernatant containing scFv or phage
antibodies were applied. Bound scFvs were detected by HRP-labeled mouse
mAb P5D4 and bound phages by HRP-labeled anti-M13 mouse mAb using 3,3',5,5'-tetramethylbenzidine (TMB, Sigma). Adding H2SO4 after 20 min
stopped the reaction, and the optical density was measured at
A450 nm (Titertek Multiskan
MCC/340).
Epitope recognition of Pfs48/45 by phage or soluble scFv was carried
out by a competition ELISA as described previously (10). Briefly,
Pfs48/45 was captured from antigen extract in microtiter plates. After
washes with PBS, wells were incubated with a mixture of 30 µl of test
sample and 30 µl of HRP-labeled anti-Pfs48/45 mAb (recognizing
various epitopes of Pfs48/45) for 120 min and detected using
3,3',5,5'-tetramethylbenzidine as described above.
Competition of (a) scFv with phage antibodies or
(b) scFv with serum antibodies from a malaria patient was
done as follows. After incubation with antigen, the wells were
incubated with: (a) 30 µl of periplasmatic scFv antibodies
(1:2 diluted with PBS containing 0.1% milk) and 30 µl of phage
antibodies (1:3 diluted with PBS containing 0.1% milk) for 120 min;
and (b) 30 µl of serum dilutions (ranging from 1/20 to
1/10, 240 diluted with PBS containing 0.1% milk) and 30 µl of
soluble scFv antibody fragments (1:2 diluted with PBS containing 0.1%
milk) for 120 min. Bound scFvs were detected as described above.
Sequencing--
The sequencing of selected clones was carried
out using the CEQ Dye Terminator Cycle sequencing kit (P/N 608000, Beckman), and the products were analyzed on a CEQ 2000 Dye Terminator
Cycle sequencer (Beckman). The sequences of VH and
VL genes were compared with the sequences present in the V
BASE Sequence Directory (17) to determine the closest germ-line counterpart.
Immunofluorescence Assay (IFA)--
An indirect IFA was done
with a mix of cultured asexual and sexual stage parasites (NF54 isolate
of P. falciparum) air-dried on multispot slides and
incubated with 20 µl of culture supernatant (1:2 diluted with PBS)
for 30 min. The slides were rinsed with PBS and incubated with 20 µl
of mAb anti-VSV-G (P5D4) (10 µg/ml in PBS) for 30 min. After
being washed with PBS the slides were incubated with AlexaTM-conjugated
goat anti-mouse IgG (Molecular Probes; diluted 1:200 in PBS containing
0.05% Evans Blue) for 30 min. The slides were rinsed, washed, mounted
with a mixture of 90% glycerol and 10% Tris-HCl (pH = 9.0) under
a coverslip, and examined under ultraviolet illumination with a Leitz
microscope. Specific green fluorescence of sexual stage parasites was
scored as a positive reaction.
For surface IFA analysis, gametocytes (see above) were allowed to
undergo gametogenesis for 30 min by resuspension at a 10% hematocrit
in fetal calf serum at 27 °C. 108 live gametes were
mixed with 10 µl of packed normal human erythrocytes/ml of PBS. From
this suspension, 106 gametes (100 µl) were incubated with
100 µl of bacterial culture supernatant containing scFv antibody
fragments for 30 min, washed with PBS, and incubated for a further 30 min with 25 µl of the anti-VSV-G mAb P5D4 (10 µg/ml in PBS).
After washing with PBS, the gametes were incubated with 25 µl of
AlexaTM-conjugated goat anti-mouse IgG (Molecular Probes; diluted 1:200
in PBS containing 0.05% Evans Blue) for 20 min. After washing the
gametes with PBS and resuspending them in 50 µl of PBS,
the fluorescence of the membrane of intact gametes was observed under
UV illumination at a magnification of ×400.
Blotting Procedures--
For an indication of scFv expression
levels in bacterial culture supernatants, nitrocellulose filters were
placed in a dot-blotting apparatus (Schleicher & Schuell), and
bacterial culture supernatant was applied. Filters were dried and
blocked with PBS containing 5% milk for 20 min, bound scFv was
detected using anti-VSV-G and alkaline-phosphatase-conjugated
rabbit anti-mouse (Dakopatts; 1:1,000 dilution). Dots were visualized
using nitro blue tetrazolium/bromochloroindolyl phosphate
(NBT/BCIP).
For the detection of scFv binding to Pfs48/45 on Western immunoblots,
gametocyte extract was fractionated on SDS-polyacrylamide gels (Novex;
NuPAGETM 4-12% Bis-Tris gel) under nonreducing conditions conducted
by Laemmli's procedure (18) at 200 V for 35 min. Molecular sizes were
estimated with SeeBlueTM Pre-stained Standard (Novex). Proteins were
electroblotted to polyvinylidene difluoride membranes in NuPAGE
transfer buffer (Novex) for 60 min at 30 V. Nonspecific binding sites
of membrane strips used were blocked with PBS containing 0.1%
milk. After primary antibody incubation, the strips were washed
with PBS and incubated with HRP-labeled mAb P5D4 (2 µg/ml in PBS
containing 0.05% Tween 20 and 0.1% milk). Strips were washed again
with PBS and developed with 3,3'-diaminobenzidine (Sigma).
Two combinatorial human scFv antibody libraries were constructed
using B cells from P. falciparum gametocyte carriers
essentially as described by Hoet et al. (9). After a primary
and a second amplification of heavy and light chain genes using PCR and
digestion of these products, both VH and VL
genes were purified by Wizard PCR prep (Promega) and ligated
sequentially in the pHENIX vector. The diversity of the resulting
libraries was more than 108 individual clones (Table
I). Library quality was analyzed
by PCR for full-length inserts (>75%) and dot-blot analysis for
percentage of clones producing soluble antibodies (Table I).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C until used.
20 °C until used for Western blot analysis, for
immobilization of Pfs48/45 in the phage selection procedure, or in ELISAs.
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Characteristics of the immune combinatorial human scFv antibody
libraries
-D-galactopyranoside induction.
The phage libraries were incubated with a Nonidet P-40 extract of gametocytes immobilized to the wall of immunotubes. After washing, the bound phages were eluted by competition with a mixture of four rat mAbs recognizing epitopes I, IIb, III, and V of Pfs48/45 at a concentration of 60 µg/ml each. Displaced phages were used for infection of bacteria, grown, combined, and subjected to further selection rounds (in a total of three rounds). After the second and third rounds, phagemid particles were precipitated by polyethylene glycol. No reactivity was found in the Pfs48/45-specific ELISA before selection. The enrichment of the anti-Pfs48/45-specific phages during selection was from an OD value of 0.09 before selection up to OD = 0.97 after three rounds of selection for the Spa library and from OD = 0.10-0.67 for the Cam library as detected in the Pfs48/45-ELISA. The phage concentration applied was the same for each biopanning (1012 colony-forming units/ml). The number of phages bound to the antigen increased during selection from 0.9 to 6.2 × 107 colony-forming units/ml for the Cam library and from 5.3 to 42.0 × 107 colony-forming units/ml for the Spa library.
After the third round of selection, 96 clones from each library were
analyzed by PCR for full-length inserts, and
isopropyl-1-thio--D-galactopyranoside induction was
performed to obtain soluble scFv expression for ELISA analysis. 53% of
the clones from the Spa library and 60% of the Cam library expressed
soluble scFv as analyzed by the dot blot technique. The percentage of
clones after the third selection with full-length inserts was 33% for
both libraries (data not shown). 30 ELISA-positive clones from each
library were subjected to fingerprinting. The Spa library gave eight
different fingerprint patterns, whereas the Cam library yielded six
different fingerprint patterns, which differed from those obtained from
the Spa library (data not shown). The positive clones (from both
libraries) in the competition-ELISA competed for epitope III of
Pfs48/45, whereas no competition was found for epitopes I, IIb, and V
of Pfs48/45 (data not shown).
Eleven clones selected from the two human libraries were further
analyzed by sequence analysis (Fig. 1) and
grouped as depicted in Table II. A
comparison with the sequences of germ-line VH genes shows
that the clones use a VH1, VH3, or
VH4 family-derived germ-line segment. Alignment with the
VL germ-line sequences showed that these clones use a
VL2 or VKI family segment. Most mutations are found in the clones derived from germ-line gene
DP15 of the VH1 family.
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These clones were analyzed further by Western blot analysis,
immunofluorescence assay, and competition ELISA. Western blotting was
performed with soluble scFv using sequential Nonidet P-40 and SDS
extracts from gametocytes. Six clones of the Spa library and two clones
of the Cam library stained the typical Pfs48/45 bands doublet, whereas
one clone of the Cam library weakly stained a 230-kDa protein band
(Fig. 2). Two clones (SG3 and KH9) that were
negative on Western immunoblot were also negative in the competition
ELISA (Table II). Four clones (SF3, SF5, SG10, and SB12) were selected
from the Spa library recognizing Pfs48/45 and an additional 30-kDa
protein, whereas clones KB6 and KG2 without the 30-kDa reaction on
immunoblot were selected from the Cam library. Clones SC8 and SF1 with
a weak reaction on immunoblot are both members of the VL2
family with different mutations compared with the germ-line gene
DPL11.
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Antibody reactivity against sexual stage parasites of isolate NF54 was determined by immunofluorescence analysis. Clear green fluorescence of the membrane of intact live gametes (surface-IFA) was seen for the clones positive for Pfs48/45 on the Western immunoblot, whereas clone KC4 (weak positive for Pfs230 on immunoblot) was negative in both the IFA and the surface IFA (Table II).
Six clones from the Spa library and two clones of the Cam library
showed significant competition for epitope III of Pfs48/45 in the
competition ELISA, whereas no competition was found for epitopes I,
IIb, and V of Pfs48/45. All clones positive in the competition ELISA
were positive for Pfs48/45 on the Western immunoblot (Fig. 2). Clone
SG10 has a weak reactivity in the competition ELISA and also a weak
reactivity in the IFA (Table II). The competition ELISA-positive clones
and the uniformity of the VH and VL genes encoding the different scFvs suggested that these clones were directed
against the same or related epitopes of Pfs48/45. To explore this
possibility, we produced scFv and phage antibodies from 11 clones to
test the capacity of phages to replace scFvs after binding to Pfs48/45.
The percentage inhibition of binding of scFvs to Pfs48/45 by phage
antibodies of clone SB12 is depicted in Fig.
3. The percentage inhibition of scFvs binding
to Pfs48/45 by phage antibodies from clones KC4, SG3, and KH9 was
comparable (data not shown). Phages of clone KC4 did only compete with
bound scFv of KC4, and no competition was found with the other clones. The same pattern was also seen for clone SG3 and KH9. Clones
SB12 to SG10 showed competition, suggesting specificity for
epitope III of Pfs48/45, whereas the clones for Pfs48/45-unrelated
antigen did not compete. These results show that phage antibodies from the eight competition ELISA-positive clones (Table II) were capable of
inhibiting the interaction of scFv antibody fragments to Pfs48/45 by
more than 65%. Collectively, these experiments support the notion that
all clones are directed against the same or closely spaced
epitopes.
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The serum of the malaria patient from which the immune library (Spa)
was constructed was tested for its ability to compete with
anti-Pfs48/45 antibody fragments for binding to Pfs48/45 in comparison
with a blood bank donor serum. The patient serum was able to inhibit
the binding of scFvs from clones KG2, SB12, and SF5 to Pfs48/45,
whereas the serum from the negative blood bank donor was unable to
inhibit binding (Fig. 4). The patient serum
competes strongly with the three clones, starting inhibition at a
reciprocal dilution of 320 for clone SF5, 640 for clone KG2, and 1280 for clone SB12. The other clones (SF3, KB6, SF1, and SC8) gave
comparable results (data not shown). Clone SG10, with the lowest
competition titer for epitope III of Pfs48/45 (Table II) started
inhibition at a reciprocal dilution of 80.
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Furthermore, 48 serum samples of gametocyte carriers were analyzed in the competition ELISA with mAb 85RF45.3 and scFv clone SB12 as competitor. 16 (33%) of 48 gametocyte carriers were able to compete with both labels in the Pfs48/45 competition ELISA at dilutions varying from 1/20 to 1/640 (Table III). Five (10%) serum samples were positive with mAb 85RF45.3 as competitor but negative with clone SB12 in the competition ELISA. The low titer serum samples (>1/160) seem to be lower with clone SB12 as competitor in comparison with the mAb 85RF45.3. These results imply that the antibody fragments selected from the two different libraries recognize similar epitope regions on Pfs48/45 as anti-Pfs48/45 antibodies present in malaria patients' sera.
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DISCUSSION |
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In the present study we employed a novel strategy for generating recombinant human monoclonal antibody fragments from peripheral B cells from malaria immune patients. The selection of recombinant human monoclonal antibody fragments specific for the Pfs48/45 antigen from phage display libraries was carried out successfully by competitive elution with mAbs specific for this antigen. This competitive elution technique between a mAb and phage antibody for binding an epitope is based on the principle of a Pfs48/45 two-site ELISA (10).
The libraries from malaria patients used in this study had at least 108 independent clones with >75% full-length insert ratios. Despite the mix of four mAbs (recognizing epitopes I, IIb, III, and V), used for competitive elution, only phage antibodies directed to epitope III of Pfs48/45 were selected. However, the serum of the malaria patient of library SpA reacted with all epitopes of Pfs48/45 as measured by the competition ELISA with the anti-Pfs48/45 rat and mouse mAbs (data not shown).
ScFvs derived from all clones of a VH1 germline gene inhibited binding of rat mAb 85RF45.3 recognizing epitope III of Pfs48/45, whereas scFvs of VH3 and VH4 families did not influence binding in competition ELISAs nor reactivity in the immunofluorescence assay. Interestingly, clone KH9 was negative in the competition and immunofluorescence assays, whereas clone SC8 was positive in these tests. They are both members of the same VL2 family but differ in their VH region (clone KH9 uses a VH4 and clone SC8 uses a VH1 family segment). The same pattern can be seen for clone SG3 (negative in different tests) and clones KG2 and KB6 (positive in different tests). They use the VKI family segment but differ in the VH region. Most likely, the reactivity to Pfs48/45 is not dependent on the VL gene. The selected scFv recognize the same epitope III but differ in their amino acid sequence and possibly in the fine specificity of their interaction with this epitope.
We have previously described (11) a fair agreement between transmission-blocking activity using a feeder assay with NF54 P. falciparum parasites and reactivity in Pfs48/45 competition ELISAs using mouse mAbs in sera from gametocyte carriers from Cameroon. These data show that overall the C45 ELISA for epitope III is a good marker for a comparison with transmission-blocking activity in serum samples. Also, Table III shows a good correlation between reactivity of scFv with the sera from gametocyte carriers in comparison with the rat mAb 85RF45.3. However, transmission-blocking activity was found only for mouse mAbs 82C4.A9 and 81D3.D2 (both are epitope III-specific and of the IgM isotype) (5). All other mouse and rat mAbs against epitope III of Pfs48/45 with an IgG isotype show no transmission-blocking activity (5, 6). In a pilot experiment, four scFv clones did not block the transmission of the parasite (data not shown). For a comparison with the blocking mouse mAbs, it is better to make Fab fragments, diabodies, or tetrabodies. In further studies, the transmission-blocking capacity of the selected clones will be studied by making these fragments. Also different selection methods will be performed to get scFvs to other epitopes.
For Pfs48/45 vaccine development, it is important to study anti-Pfs48/45 antibody profiles in endemic sera after natural infections. So far, the possibility for such study was limited because of high background reactivity when using mouse and rat mAbs to capture antigen. This problem could be resolved in part by using F(ab')2 fragments; however, different methods failed so far to produce and purify F(ab')2 fragments of these anti-Pfs48/45 mAbs. Because scFvs lack the cross-reactive mouse and rat antibody domains, we anticipated that high background could be avoided. Preliminary data suggest that the background can be circumvented by these scFv preparations.
In conclusion, the successful selection of human scFv against Pfs48/45
paved the way for future selection of more recombinant antibodies with
possible transmission-blocking activity. This option would
emphasize the importance of this molecule as a vaccine candidate.
Malaria patient-derived phage antibody display libraries can thus be
used to isolate specific immunological reagents of human origin, which
is useful in both the characterization of the parasite's antigenic
composition and the human hosts' immune response to malaria infection.
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ACKNOWLEDGEMENTS |
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We are grateful to Marga van de Vegte-Bolmer for parasite cultures.
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FOOTNOTES |
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* The Dutch Ministry for Development Co-operation (DGIS/SO) Contract NL002701 supported this research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A research fellow of the Royal Netherlands Academy of Arts and
Sciences (KNAW).
§ To whom correspondence should be addressed: UMC St. Radboud, Dept. of Medical Microbiology, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31-243619186; Fax: +31-243614666; E-mail: W. Roeffen@mmb.azn.NL.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M100562200
2 J. M. H. Raats, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: mAb, monoclonal antibody; scFv, single chain variable fragment; VL, variable light chain: VH, variable heavy chain; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; IFA, immunofluorescence assay; VSV-G, vesicular stomatitis virus glycoprotein.
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REFERENCES |
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