Journal of Immunological Methods 294 (2004) 199 207

www.elsevier.com/locate/jim

Protocol

A simple and rapid protocol for the sequence determination

of functional kappa light chain cDNAs from
aberrant-chain-positive murine hybridomas
Xin Yuana, Michael J. Gubbinsb, Jody D. Berrya,c,*
a
The Monoclonal Antibody Section,
National Microbiology Laboratory-Health Canada and the National Centre for Foreign Animal Disease-CFIA,
1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3M4
b
The National Microbiology Laboratory, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3M4
c
Department of Immunology and the Department of Medical Microbiology and Infectious Diseases, Basic Medical Sciences Building,
The University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
Received 1 July 2004; received in revised form 1 September 2004; accepted 2 September 2004
Available online 4 October 2004

Abstract

This protocol describes the application of a polymerase chain reaction to allow the cloning and sequencing of new functional
kappa light chain cDNAs from murine hybridomas co-expressing aberrant endogenous kappa chain mRNAs. The presence of
kappa light chain aberrant mRNAs can hinder or even prevent determination of the sequence of functional murine kappa light
chain cDNAs amplified by PCR from hybridomas. The method described here employs a panel of kappa primers in the presence of
molar excess of a primer complementary to the complementary determining region (CDR) 3 of the known aberrant chain sequence.
Analysis of the PCR products reveals two bands for some reactions: one the functional, full-length kappa chain cDNA (~400 bp)
and another shorter (~100 bp) band corresponding to short aberrant chain kappa CDR3-constant region. The full-length product is
gel purified and cloned prior to sequencing and aligned with V-region germline sequences available in NCBI and GenBank
databases. This method is used routinely in our laboratory and demonstrates consistency and reliability for sequence determination
of kappa light chain V-gene cDNA of mAbs to diverse antigens. This protocol is a rapid and convenient method for determining the
sequence of murine V kappa region genes from hybridomas expressing aberrant kappa chain mRNAs.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Immunoglobulin; Variable gene; Sequencing; Hybridomas; Rapid

Abbreviations: CDR, complementary determining region; FR1, framework region; AbVk, aberrant endogenous myeloma kappa chain; RT
PCR, reverse transcriptionpolymerase chain reaction; Vk, light chain variable domain, comprising the V and J segments; VH, heavy chain
variable domain, comprising the V, D, and J segments.
* Corresponding author. The Monoclonal Antibody Section, National Microbiology Laboratory-Health Canada and the National Centre for
Foreign Animal Disease-CFIA, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3M4. Tel.: +1 204 789 6063; fax: +1 204 789 2038.
E-mail address: berryjd@inspection.gc.ca (J.D. Berry).

0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2004.09.001
200 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207
1. Background
The V-domain is responsible for determining the
binding specificity of an antibody molecule. The
immunoglobulin V-domain is encoded by minigene
elements recombined at the DNA level during B
cell development in the bone marrow (Tonegawa,
1983). Somatic DNA recombination events, involv-
ing a complex series of enzymes (Bassing et al.,
2002), bring together a VH, DH, and JH gene
segment for the H chain, and Vn and Jn or VE
and JE gene segments for the L chain (Janeway et
al., 1999; Tonegawa, 1983) to encode a functional
heavy and light chain variable region, respectively.
The germline, junctional and combinatorial diversity,
together with antigen dependent changes such as
hypermutation and receptor-editing with B cell
selection, collectively produce a potentially vast
diversity of V-gene sequences. The molecular
identity of pathogen specific V genes in new
hybridomas is unknown and must be determined
empirically via cloning and sequencing. The V gene
sequence represents the primary sequence encoding
antigen binding specificity. This data is useful for
studying V-gene immunogenetics and more impor-
tantly is required for the construction of recombi-
nant antibody for exploitation in therapy and
diagnostics. The sequence information is also
important for patent disclosure and clone identity.
Therefore, the nucleic acid sequence encoding the
V-domain is of considerable importance.
Hybridomas, first developed by Kohler and Mil-
stein (1975), are immortal cell lines that produce
mAbs. Hybridomas are a valuable cellular resource
for generating recombinant mAbs (monoclonal anti-
bodies). Endogenous properties of hybridoma cell
lines can hinder recombinant mAb development.
Commonly used myeloma fusion partners such as
Sp2 and p3-X63-Ag8.653 were derived from the
MOPC-21 myeloma line (Leahy et al., 1988; Carroll
et al., 1988, respectively). The p3-X63-Ag8.653
myeloma line is widely used in both industry and
academia for hybridoma development from immu-
nized mice. The majority of myeloma cell lines are
derived from the MOPC-21 clone which expresses an
endogenous and aberrant Vk mRNA. This mRNA is
not translated into a fully functional IgG protein due
to the presence of a four nucleotide deletion at the
light chain VJ joining site which produces a stop
codon immediately following complementary deter-
mining region (CDR) 3, thereby preventing trans-
lation of the kappa constant domain (Carroll et al.,
1988). The majority of hybridomas derived from SP2
or p3-X63-Ag8.653 myelomas have a functional
heavy chain transcript and two possible light chain
transcripts; one functional, and one aberrant. The
presence of all three of these transcripts indicates that
the cell line did not lose the chromosomes from the
parental B cell which encode the heavy and light
chain (6 and 12, respectively), as well as the
endogenous chromosome 12 of the myeloma that
encodes the aberrant kappa chain mRNA, during the
stabilization process inherent in hybridoma fusion. In
some cases the aberrant kappa chain gene may be
either silenced or lost from the multiploid hybridoma
line during stabilization, as many p3-X63-Ag8.653
derived hybridomas do not express the endogenous
kappa chain mRNA.
The polymerase chain reaction is widely used to
amplify and clone functional V-gene cDNAs from
hybridomas. Importantly, degenerate and consensus
primers designed to amplify kappa light chain V-
gene cDNA from kappa-isotype hybridomas will
amplify the aberrant chain, if it is present, as the
primers cannot distinguish the aberrant from the
functional cDNA. In many cases the aberrant chain
is not problematic, and the chromosome may have
been lost during cell stabilization and aminopterin
selection following fusion. PCR techniques such as
RACE PCR (rapid amplification of cDNA ends)
have been used to clone V-genes when they are
difficult to amplify (Ruberti et al., 1994). However,
these techniques do not favor the amplification of
functional Vk. We have developed a primer-com-
petition method which instills a bias in the PCR
procedure to favor amplification of full-length
functional kappa chains. This simple procedure
dramatically reduces the aberrant variable kappa
chain background and enhances the amplification
and recovery of functional kappa chain cDNA. A
molar excess of an oligonucleotide primer is
included in the PCR mixture along with the panel
of 5Vupstream framework region (FR) 1 primers and
with the kappa constant region 3V primer. We
reasoned that this will bias the PCR and result in
the formation of two products; the truncated
 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207
aberrant chain cDNAs, and the full-length functional
kappa V-gene cDNA.
2. Type of research
The construction of recombinant monoclonal anti-
body from hybridoma expressed monoclonal anti-
bodies requires the cloning and assembly of
immunoglobulin variable region cDNAs (V-gene
cDNA) from hybridomas.
(a) Immuno-genetic study of structure and biolog-
ical function of mAbs.
(b) Cloning on phage for in vitro affinity matura-
tion/improvement studies and expression of
recombinant mAbs.
(c) Cloning on phage for in vitro humanization
studies.
3. Time required
(1) Hybridoma cell collection (30 min).
(2) RNA isolation (30 min).
(3) PCR first panel (4 h).
(4) PCR with AbVk (step 3 can be omitted and step
4 done preemptively).
(5) Cloning of products (30 min and growth
overnight).
6) Pick plated clones, grow cultures overnight and
perform plasmid miniprep purification (10
clones, grow overnight, 1.5 h to purify and send
for sequence analysis).
4. Materials
4.1. Reagents
4.1.1. Hybridoma growth, RNA purification
! BD cell mAb medium (BD, San Jose, CA).
! PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g
NaH2PO4, and 0.24 g KH2PO4 in 1 l H2O,
pH 7.4).
! Trypan blue (Sigma, St. Louis, MO).
! Rneasy total RNA isolation kit (Qiagen, Germany).
201
4.1.2. Reverse transcriptionpolymerase chain reac-
tion (RTPCR)
! RT buffer (250 mM TrisHCl pH 8.3, 375 mM
KCl, 15 mM MgCl).
! Superscript II First strand cDNA kit (Invitrogen,
Canada).
! 10 Taq DNA polymerase buffer (Invitrogen).
! 100 mM dNTPs solution (Invitrogen).
! 50 mM MgCl2 solution (Invitrogen).
! Taq DNA polymerase enzyme (Invitrogen).
! 20 pmol Oligonucleotide primers (Invitrogen).
! Ultra Pure Agarose (Invitrogen).
! 100 bp DNA ladder (Invitrogen).
! Low DNA mass ladder (Invitrogen).
! QIAquick gel extraction kit (Qiagen).
! QIAquick PCR purification kit (Qiagen).
! ABI PRISM Dye Terminator Cycle Sequencing
Kit (Perkin-Elmer, Norwald, CT).
5. Special equipment
! Techne Gradient Thermocycler (Rose Scientific,
Canada).
! Automated DNA sequencer.
6. Detailed procedure
6.1. Hybridoma cell collection
The hybridomas were developed in house using the
p3-X63-Ag8.653 myeloma cell line and methods
essentially as described previously (Berry et al.,
2004). We have applied the protocol to five hybrid-
oma cell lines which were discovered to express the
MOPC-21 aberrant kappa light chain mRNA. Total
cellular RNA was harvested from the hybridomas
using methods essentially as reported previously
(Gubbins et al., in press). Clone F24G2 expresses an
IgG3/k mAb against FMD virus. Clones F20G1,
F20G6, and F20G8 express IgG1/k mAbs to the
Bacillus anthracis protective antigen (PA). Hybrid-
oma 2D5 was kindly provided by Dr. Stephen Little
(USAMRIID, Fort Detrick, MA). The 2D5 hybridoma
was made from a fusion of the SP2/0-Ag14 myeloma
to immune splenocytes from mice immunized with
202 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207

purified protective antigen from culture of B. anthra- Table 1

cis (Little et al., 1988). Viable, log-phase cells were Light chain variable region oligonucleotide primers
counted (Trypan blue exclusion principle) and 25 Name Sequence
million cells were collected as follows. MKcL-3 5V 5V TTG CTG TTC TGG GTA TCT GGT A 3V
UMIgVK 5Va 5V GAC ATT CTG ATG ACC CAG TCT 3V
MVL 5V 5V GTG CCA GAT GTG AGC TCG TGA TGA
(1) Collect enumerated (110 million) viable cells
CCC AGT CTC CA 3V
by centrifugation at 285g for 8 min in a Sheriff 5V GCC GAA TTC GAC ATT GTG CTG
sterile 50-ml conical tube (Falcon) and resus- Kappa5Vb ACC CAA TCT CCA GCT TC 3V
pend in 40 ml of fresh medium. This step must Sheriff 5V GCG AAT GCG GAT GTT AAC TGC
be done immediately after counting the cells as Kappa3Vb TCA CTG GAT GGT GGG 3V
IgKappa 3V 5V GCG CCG TCT AGA ATT AAC ACT CAT
the cells must be viable for quality RNA to be
TCC TGT TGA A 3V
harvested. MKC1 3V 5V GGA TAC AGT TGG TGC AGC 3V
(2) Spin down to a pellet once again as above. MK-3Seq 3V 5V TAC AGT TGG TGC AGC ATC AGC 3V
Resuspend cells in residual medium by tap- AbVK 5V 5V ACC TAT TAC TGT CAG CAC ATT A 3V
ping the conical tube on the flow hood a
Dattamajumdar et al. (1996).
b
surface. Bossart-Whitaker et al. (1995).
(3) Add 200 Al of RNA lysis buffer and mix
gently but thoroughly and perform the RNA 1 Al of AbVK (175 pmol/Al)
isolation procedure as per commercial instruc- 0.5 Al of Taq DNA polymerase enzyme
tions (Qiagen). (2.5 units).
(4) Use 2 Ag of total or purified mRNA per cDNA (4) Mix the reaction tube well by tapping on the side
reaction performed using Superscript II RT kit of the tube and place it on ice.
(Invitrogen) as per commercial instructions. (It (5) PCR conditions were 94 8C for 2 min followed
is acceptable to stop and store the cells once by 30 cycles (94 8C for 1 min, 62 8C for 1 min,
lysed in RNA lysis buffer (at 80 8C) or at the 72 8C for 1.5 min) and the final extension step of
purified RNA step for the short term, however, it 72 8C for 10 min.
is recommended that the RNA first be converted (6) After the reaction, the samples were electro-
into the more stable cDNA form prior to phoresed on a 1% TAE (TrisacetateEDTA)
storage). agarose gel for 1 h at 90 V.

6.2. PCR with panel of Vk primers
A list of oligonucleotide primes is given in Table 1
and their respective binding sites are depicted in Fig. 1.
PCR reaction:
(1) Aliquot 50 ng of the first strand cDNA into a
0.6-ml microcentrifuge tube, add sterile distilled
water to adjust the volume to 10 Al.
(2) Place the reaction tube on ice.
(3) Add the other components required for the PCR
into the reaction tube in the following order:
24.5 Al of sterile distilled water
5 Al of 10 Taq DNA polymerase buffer
5 Al of 2.5 mM dNTPs
2 Al of 50 mM MgCl2
1 Al of 5V primer (20 pmol/Al)
1 Al of 3V primer (20 pmol/Al)
6.3. Extraction of the gel-purified (~400 bp) DNA
fragments and cloning of the isolated immunoglobulin
kappa chain variable region gene into the Topo-TA-
cloning vectorR
Materials:
(1) Full-length kappa chain immunoglobulin vari-
able region DNA fragment from the agarose
gel.
(2) QIAquick Gel Extraction Kit (Qiagen Cat.
No. 28706).
(3) Topo-TA-cloning vectorR (PCR 2.1-TOPO)
vector 10 ng/Al plasmid DNA (Invitrogen
Cat # 46-0811).
(4) 6 TOPO Cloning Stop Solution (Invitrogen,
Cat # 46-0066) (transformation and plating as
per commercial instructions).
 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207 203

Fig. 1. Flowchart depicting the strategy designed to successfully determine the sequence of functional kappa variable region genes from murine
hybridomas made from fusions with p3-X63-Ag8.653 myelomas. (1) Hybridomas are used as a source of immunoglobulin heavy and light chain
RNA and reverse transcribed to make first strand cDNA. (2) A panel of primers is used to amplify murine heavy (not shown) and light chain
cDNAs using polymerase chain reaction. Correctly sized products are cloned and sequenced. (3) If a functional light chain cDNA is found in the
majority of clones sequenced, the sequence determination is complete. If the sequence is found to be aberrant chain, continue on to step 4. (4)
Repeat initial panel of primers in PCR with additional primer complementary to the AbVk light chain CDR3 in molar excess to force truncation
of the aberrant cDNA product and bias amplification of full-length functional light chain (~350400 bp). The inset gel lanes are as follows:
(MW) 100 bp ladder (Invitrogen). (1) Primer pair MkCL-1 and MkC1 with Abvk. (2) MkCL-2 and MkC1 with AbVk. (3) MkCl-3 and MkC1
with AbVk. (4) MkCl-4 and MkC1 with AbVk. (5) UmIgVk and MkC1 with AbVk. (6) UmTgVk and Mk3-seq with AbVk. (7) Sherrif 5-kappa
and MkC1 with AbVk. Lastly, the correct sized products are cloned and sequenced to obtain the full-length functional kappa light chain
sequence.
204 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207
Procedure:
(1) Excise the 400 bp cDNA fragment from the
agarose gel with a clean, sharp scalpel.
(2) Weigh the gel slice in a microcentrifuge tube.
Add 3 vol of Buffer QG to 1 vol of gel and purify
as per commercial instructions and elute with 50
Al of sterile distilled water.
(3) Ligate the cDNA into commercial Topo-TA-
cloning vectorR (PCR 2.1-TOPO) as per manu-
facturers instructions.
6.4. Method for colony screening by PCR
Materials:
(1) Sterile distilled water.
(2) Bacterial clones.
(3) 10 PCR buffer (Invitrogen).
(4) 100 mM dNTPs solution (Invitrogen).
(5) 50 mM MgCl2 solution (Invitrogen).
(6) Taq DNA polymerase enzyme (Invitrogen, Cat.
No. 10966-034).
(7) 20 pmol flanking oligonucleotide primers
(Invitrogen).
Procedure:
Sample preparation:
(1) Place a single bacterial colony in 50 Al sterile
water in a 0.6 ml microcentrifuge tube.
(2) Vortex briefly to resuspend the bacteria.
(3) Heat the tube to 100 8C for 5 min to lyse the
bacteria and inactivate endogenous nucleases.
(4) Cool on ice for 3 min.
(5) Spin the bacterial debris down in the micro-
centrifuge at 13,000 rpm for 2 min.
(6) Take 10 Al supernatant for PCR template and
place it in a new 0.6-ml microcentrifuge tube
without disturbing the pellet.
PCR reaction:
(1) Prepare master mix per reaction in a 2-ml
microcentrifuge tube based on the following
volumes per reaction:
24.5 Al of sterile distilled water
5 Al of 10 Taq DNA polymerase buffer
5 Al of 2.5 mM dNTPs
2 Al of 50 mM MgCl2
1 Al of T7 or M13 reverse primer (20 pmol/Al)
0.5 Al of Taq DNA polymerase enzyme
(2) Mix well by pipetting up and down and place on
ice.
(3) Heat the 10 Al DNA template (above) to 94 8C
for 5 min to denature.
(4) Cool on ice for 5 min.
(5) Briefly spin the denatured template in the
microcentrifuge to bring all 10 Al to the bottom
of the tube.
(6) Add the master mix (40 Al) into the DNA
template tube. Pipette up and down to thor-
oughly mix the reaction mixture.
(7) Run the program: PCR condition were 94 8C for
2 min followed by 30 cycles (94 8C for 1 min, 62
8C for 1 min, 72 8C for 1.5 min) and the final
extension step of 72 8C for 10 min.
(8) After the reaction, analyse 10 Al of sample on a
standard 1% agarose gel.
(9) Sequence more than three insert positive clones
for each light chain by automated sequencing,
from both directions.
(10) Analyse the sequence data.
7. Results
This protocol outlines a simple primer-competition
polymerase chain reaction that allows the successful
amplification and identification of novel antigen
specific murine kappa light chain cDNAs from p3-
X63-Ag8.653 derived hybridomas. A flowchart
depicting the simplified protocol for the PCR cloning
of rearranged kappa chain immunoglobulin variable
region gene cDNA from MOPC-21 endogenous
aberrant chain positive hybridomas is shown in Fig.
1. A panel of oligonucleotide primers, complementary
to kappa framework 1 (Table 1) are used in individual
PCR reactions to attempt to amplify the unknown
functional V gene cDNA. Successfully amplified
cDNAs are gel purified, cloned and sequenced. New
functional kappa chain cDNAs are distinct from the
aberrant MOPC-21 light chain cDNA and the cloning
is complete. However, if the sequence is found to be
that of the MOPC-21 aberrant chain, the modified
PCR is performed using the primer panel with the
 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207 205

AbVk primer included in the master mix. The simple

addition of a third primer (AbVk), specific to the
aberrant kappa CDR3, reduces the interference of the
endogenous aberrant kappa template sufficiently to
bias amplification of full-length functional kappa
cDNA from hybridomas. This is important in cases
where the use of degenerate or consensus primers
inherently results in competitive co-amplification of
the functional kappa cDNA and the aberrant kappa
cDNA templates which are together in all reaction
mixes (Fig. 1).
The addition of the AbVk primer short-circuits the
complete amplification of the aberrant kappa cDNA
and results in the biased amplification of full-length
functional kappa cDNA. The PCR amplification
products are again analyzed using agarose gel
electrophoresis. Reactions typically show two distinct
bands at 400 and 100 bp. In this case, the correctly
sized (~400 bp) fragment is gel purified from the
reaction products because the small band (~100 bp) is
the truncated aberrant product in that reaction (Fig.
1). The full-length products are cloned and subjected
to sequence analysis. Elucidation of kappa light chain
products, which was inhibited by the nonfunctional
MOPC-21 aberrant cDNA, is shown in Fig. 2.
This protocol allows for the determination of kappa
chain cDNAs with diverse sequences. The cloned
cDNAs were sequenced using vector specific primers.
The sequences of the five functional kappa light
chains were determined and the inferred amino acid
sequences are shown in Fig. 2. Analysis of the CDR
regions shows that diverse sequences can be obtained
by using this method. Indeed, analysis of the inferred
amino acid sequence shows that kappa domains with
1017 amino acid residues in CDR1 can be obtained
(Fig. 2). The ability to clone and sequence unknown
kappa light chain cDNAs is important for the
construction of recombinant mAbs using hybridoma
derived cDNAs.

Fig. 2. Inferred amino acid sequences of the functional kappa light

chains determined for the hybridomas in this protocol. The relative
positions of the framework regions (FR1, 2, 3, 4) and the
complementarity determining regions (CDR1, 2, 3) are shown.
The GenBank accession numbers are shown in the column on the
right and are AY631279-AY631281 (for 2D5, F20G8, F20G76,
light chains, respectively), AY631283 for F20G1 light chain, and
AY631282 for the F24G2 light chain.
206 X. Yuan et al. / Journal of Immunological Methods 294 (2004) 199207
7.1. Discussion
Competitive V-gene PCR amplification is a
convenient and rapid method to determine the
sequence of functional antibody V-genes in hybrid-
omas that express the endogenous aberrant myeloma
kappa mRNA. It avoids the added expense, time, and
technology required for more complex procedures
such as peptide-nucleic acid clamping (Cochet et al.,
1999), restriction digestion/screening (Leung et al.,
1993), or ribozyme cleavage protocols (Duan and
Pomerantz, 1994) that are not always practical in
many molecular biology laboratories. The protocol
described herein for p3-X63-Ag8.653 derived
hybridomas is only used when required as not all
hybridomas need this treatment. While the PCR
conditions are quite robust, the reaction series might
need to be optimized depending on the model of
thermocycler used, polymerase enzyme used, and the
exact sequence of the primers employed. Minor
changes in the annealing temp will likely affect the
efficiency of the reaction as for PCR amplification of
any gene.
The approach has been useful for obtaining kappa
sequences data from hybridomas derived from mainly
p3-X63-Ag8.653 MOPC-21 myelomas. The selection
of an appropriate myeloma partner for hybridoma
development is important and impacts downstream
applications. There are clearly different properties
among the murine myelomas NS-1, SP2/0-Ag14 and
p3-X63-Ag8.653, despite their common origin. For
example, NS-1 and p3-X63-Ag8.653 produce more
functional binders than SP2/0-Ag14. However, SP2/0-
Ag14 produces more total clones for the initial screen,
grows faster from single cells, and more readily
produces ascites in BALB/c mice (Berry, personal
observations).
This method resulted in the successful amplifica-
tion and sequencing of one functional kappa light
chain from a SP2/0-Ag14 derived hybridoma. The
endogenous chain bias is even higher in NS-1 clones
(data not shown) as the NS-1 myeloma cell line has
been shown previously to co-express a productively
rearranged MOPC-21 kappa gene (Walfield et al.,
1980) as well as the aberrant kappa mRNA. Clearly,
the cumulative interference of these two kappa
mRNAs would increase the bias for PCR amplifica-
tion of endogenous kappa cDNAs and interfere with
the recovery of a specific kappa mRNA sequence, and
is consistent with the enhanced transcription of the
aberrant mRNA in particular myeloma derived cell
lines (Carroll et al., 1988). Furthermore, following the
fusion process, each hybridoma undergoes chromo-
somal stabilization and loss independently in an
unexplained, and likely random, fashion. This process
could result in the loss of important enhancers or
repressors of kappa gene expression, re-organization
of chromatin patterns, or the loss of whole chromo-
somes encoding immunoglobulin genes. This experi-
ence suggests that the p3-X63-Ag8.653 myeloma is a
fusion partner of choice for scientists considering
downstream applications like recombinant mAb
development, V-gene analysis, and other in vitro
techniques (Berry, 2004).
7.2. Summary
In summary, this procedure provides a rational
approach for the isolation of functional V-gene cDNA
from the potentially diverse assortment of kappa chain
assemblages produced in murine monoclonal anti-
bodies. This will facilitate the development of
recombinant mAbs derived from murine hybridomas,
which would otherwise not be possible due to the
presence of the interfering endogenous kappa mRNA,
by quickly providing a solution to the sequence
information needed to design primers for expression
cloning.
Acknowledgements
The authors would like to thank Drs. Paul
Kitching, Frank Plummer, Raymond Tsang, and Amin
Kabani for continued support on this protocol. Thanks
enormously to Dr. Steven Little (USAMRIID) for the
generous gift of the hybridoma 2D5. This work was
funded by CRTI 0091RD, Health Canada and the
Canadian Food Inspection Agency.
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