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Department of Microbiology, Immunology, and Molecular Genetics, and Molecular Biology Institute, University of California, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or 
constant region domains and expressed
with mouse-human chimeric H chains of the four human IgG isotypes. The
resulting Abs were secreted as fully assembled molecules although, as
has been previously observed, IgG4 with either or L chains was
also secreted as HL half-molecules. However, the isotype of the L chain
can influence the kinetics of intracellular assembly with IgG1,
IgG2, and IgG4 assembling more slowly than their
counterparts. The isotype of the L chain also influenced the
susceptibility of the interchain disulfide bonds to attack by reducing
agents with variable effects, depending on the isotype of the H chains.
For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain
influenced the rate of clearance in mice, with IgG2 having a shorter
in vivo half-life than IgG2. Only slight differences were also
observed between and molecules in their kinetics of binding to
and dissociation from the hapten dansyl. These studies demonstrate that
the isotype of the L chain has only a slight impact on the structural
and functional properties of variable region identical
Abs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 
, 
, or 
with
four subclasses of and two of in the human. The L chains come
with two different constant regions, and . In the human, there
is one gene and four highly similar functional genes. The ratio
of the two types of L chains present in the serum varies from species
to species. In mice, the : ratio is 20:1, whereas in humans it is
2:1, and in cattle it is 1:20. Each chain folds into globular, 
barrel structures called domains. Both the H and L chains of the Ab
molecule are divided into variable and constant regions. The H chain
possesses one variable (VH) and three or four
constant (CH) domains depending on the Ig class,
whereas the L chain has only one variable (VL)
and one constant (CL) domain. Ab molecules are bifunctional proteins, wherein a specific recognition/binding capability is combined with "effector" functions to target and eliminate pathogens and foreign molecules. The variable regions comprise the Ab binding site and are responsible for the ability of the Ab molecule to recognize a diverse universe of Ags. It is generally accepted that the effector functions of the Ab such as activation of the complement cascade, mediation of immune phagocytosis, and Ab-dependent cell cytotoxicity are determined by the Fc portion of the molecule. Within the Fc, many of the structural motifs responsible for the recognition of effector elements have been identified (1, 2, 3, 4). The different constant regions of the H chain are known to endow the Ab with a broad range of associated functional properties. We have now addressed the question of whether the different L chain constant regions can also contribute to the structural and functional properties of the Ab molecule.
Within the IgG molecule, strong, noncovalent, lateral forces, as well as interchain covalent disulfide bonds, are essential for proper quaternary structure and function of the Ab. Longitudinal interactions between domains in native Ig molecules are minimal (5, 6, 7). Within the F(ab')2 portion of the Ab molecule, there are two structural units, VL-VH and CL-CH1. The fine structure of the VL-VH unit determines the binding specificity of the Ab. The structure of CH1 depends on the isotype of the H chain and the structure of CL depends on the isotype of the L chain. Although it is widely appreciated that the F(ab')2 determines the recognition of Ag by the Ab (8), recent evidence has suggested that the structure of the F(ab')2 may also influence functions normally assumed to be solely dependent on the Fc (9). Indeed, when the inter-H and -L chain disulfide bond pattern of IgG1 was altered to resemble that of IgG4, the ability of the mutant Ab to effect Ab-dependent cell cytotoxicity was abolished, and its ability to activate complement was reduced 15-fold (10).
We have now focused on the
CL-CH1 module within the
F(ab')2 and have produced and analyzed Abs with
or CL associated with
CH1 from the four human isotypes. All
molecules have identical
VL-VH. The isotype of the L
did not influence the ability of the Abs to activate complement and had
only a slight impact on their ability to bind Ags. However, the isotype
of the L chain did influence the in vivo half-life of IgG2, a property
assumed to be determined by the Fc of the Ab.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse anti-dansyl
(anti-DNS)4
V and VH genes from the
hybridoma 27-44 had previously been joined to human
C in the expression vector pSV184
Hneo
and to human H chain
(C1
C4) in the
expression vector pSV2Hgpt (11). To produce
the V-C molecule, the
anti-DNS V gene was joined to the human
C2 in the expression vector pAN1710.
Production of recombinant proteins
The mouse myeloma cell line NSO/1, which does not produce any
Ig, was used as a recipient for transfection of the L chain expression
vectors encoding the
V-C or
V-C proteins.
Transfection was performed as described previously (9).
Clones surviving after drug selection were screened for L chain
production by - or -specific sandwich ELISA. -Chain expression
vectors were then transfected into L chain-producing transfectants, and
surviving clones were screened for Ab production by ELISA using
DNS/BSA-coated plates. Bound recombinant Ab was detected using
polyclonal goat Ab against human IgG constant regions conjugated to
alkaline phosphatase (Sigma-Aldrich, St. Louis, MO). Ab-positive clones
were selected and subcloned. Cell lines producing the four human IgG
isotypes were identified and expanded. Selected clones were grown in
IMDM containing FCS. Culture supernatants were harvested, and
recombinant Abs were purified by affinity chromatography using
DNS-Sepharose columns as described previously (12).
Protein concentration in the purified material was estimated using a
colorimetric microassay (BCA Protein Assay; Pierce, Rockford, IL) and
was confirmed by SDS-PAGE.
Reduction-alkylation studies
Purified recombinant proteins were treated with DTT (Sigma-Aldrich) at variable molar ratios (DTT:IgG disulfides = 100:1; 20:1, 4:1, and 0.8:1). To calculate the DTT:disulfide bond ratio required for each IgG subclass, it was assumed that all cysteines are present as disulfides, and the following equations were used: (µg IgG) x (nmol IgG/µg IgG) x (nmol disulfide/nmol IgG) = nmol IgG disulfide; and nmol IgG disulfide x molar excess factor = nmol of DTT required.
After DTT treatment, iodoacetamide (Sigma-Aldrich) was added to obtain a minimum of 10-fold molar excess over total thiols in solution (including both protein thiol and reductant thiol). The following formula was used to calculate the amount of alkylating agent required: 10 (nmol DTT) x (2 nmol SH/nmol DTT) + (nmol IgG disulfide) x (2 nmol SH/disulfide)] = nmol iodoacetamide.
Protein samples (5 µg/sample) were diluted in 0.1 M Tris-Cl, pH 7, incubated in the presence of indicated amounts of DTT at 37°C for 30 min, and then cooled on ice. Iodoacetamide was then added to each sample and samples were further incubated at 37°C for 60 min in the dark. Finally, samples were placed on ice and were immediately processed for SDS-PAGE analysis.
Biosynthetic labeling and pulse-chase experiments
IgG-secreting transfectomas (
106 cells)
were washed twice in methionine-free DMEM (Irvine Scientific, Irvine,
CA) supplemented with nonessential amino acids (Life Technologies,
Grand Island, NY) and glutamine (29.2 µg/ml). Cells were labeled
overnight at 37°C in 1 ml of DMEM containing 10 µCi of Easy Tag
(New England Nuclear, Wilmington, DE). Culture supernatants and cells
were processed as described below for subsequent analysis by SDS-PAGE
and autoradiography.
For pulse-chase experiments, 107 cells were incubated at 37°C in 50 µCi/ml [35S]methionine (Amersham, Arlington Heights, IL) for 5 min. The pulse was stopped by adding 10 ml of prewarmed IMDM containing a 100-fold excess of unlabeled methionine and 10% horse serum. The material was then split into 10 aliquots of 1 ml each and was incubated for the indicated times at 37°C. Then, 1 ml/sample of ice-cold IMDM was added and the cells were harvested onto ice and collected by centrifugation. Supernatants were transferred to clean polypropylene tubes and the cell pellets were lysed in 0.5 ml of NDET (1% Nonidet P-40, 0.4% deoxycholate, 66 mM EDTA, 10 mM Tris, pH 7.4) and 0.06 M iodoacetamide. Soluble material was separated by centrifugation and transferred to clean tubes. Secreted or cytoplasmic Ig was immunoprecipitated by incubation with a mixture of rabbit anti-human F(ab')2 and Fc antisera followed by a preparation of Staphylococcus aureus protein A (IgGsorb; The Enzyme Center, Boston, MA). The precipitates were washed twice with NDET containing 0.3% SDS and once with dH2O, resuspended in sample buffer (25 mM Tris, pH 6.7, 2% SDS, 10% glycerol, and 0.008% bromphenol blue), boiled, and analyzed by SDS-PAGE and autoradiography.
Metabolic clearance experiments
Metabolic clearance for each of the recombinant proteins was studied in 8-wk-old, female BALB/c mice (groups of three mice per recombinant protein). One week before administration of radioiodinated IgGs, drinking water was supplemented with 0.1 mg/ml potassium iodide (KI) to block thyroid uptake of radioactive iodine. Radioiodination of IgG was performed using Iodobeads (Pierce) following the manufacturer’s recommendations. Na125I (100 µCi) was incubated for 5 min at room temperature with one Iodobead, and then 25 µg of IgG was added. After a 10-min incubation, the iodinated protein was transferred to a new vial to stop the reaction. Radiolabeled IgGs were separated from free iodine by size exclusion chromatography on a 10-ml Sephadex G50 column (Pharmacia, Piscataway, NJ). Labeling efficiency was estimated by determining TCA-precipitable radioactive counts and was consistently greater than 96%. SDS-PAGE and autoradiography of labeled material also confirmed that it was intact (data not shown).
Mice were injected with 5–7 x 105 TCA-precipitable cpm of labeled Ab in 100 µl of PBS by tail-vein injection, and whole-body activity was recorded using a whole-body counter. Counts were registered at the indicated times, corrected for background activity and isotopic decay, and used for production of clearance curves and calculation of Ab half-life.
Plasmon surface resonance analysis of Ag binding
Plasmon surface resonance analysis was performed essentially as
described (9). Briefly, commercially available IAsys
cuvettes precoated with high m.w. carboxymethylated dextran (Fisons
Applied Sensor Technology, Cambridge, U.K.) were used to immobilize the
Ag, a DNS-BSA conjugate with a DNS:BSA ratio of 19:1, following the
manufacturer’s instructions. The covalently bound DNS-BSA increased
the sensorgram response by 5500 arc seconds, indicating that the
cuvettes were coated with 4 x 1012
DNS-BSA molecules. For Ab binding studies, the Ag-coated cuvette was
washed with PBS and Tween 20 (PBST; 20 mM NaPO4, 0.85% NaCl,
and 0.05% Tween 20, pH 6.8), and a stable baseline was obtained before
addition of Ag-specific Ab. Abs were added to 200 µl of PBST in
volumes between 0.5 and 4 µl at time zero and association was
observed. When the association curve approximates a plateau, the Ab
solution was removed and replaced with PBST, and the dissociation
followed with time.
Direct lysis assays
Complement (C')-mediated hemolysis was performed as described previously (13). Briefly, SRBC (Pocono Rabbit Farm, Canadensis, PA) were coated with DNS-BSA by the chromic chloride method (14) and were resuspended at 0.4% in HBS-BSA (0.01 M HEPES, 0.15 M NaCl, 0.5 mM MgCl2, 0.15 mM CaCl2, and 0.1% BSA, pH 7.4). Twenty-five microliters per well of this suspension was added to round-bottom 96-well plates (Costar, Corning, NY). Recombinant IgGs in HBS-BSA at various concentrations were subsequently added in a volume of 50 µl/well and, after mixing, reactions were incubated for 15 min at 4°C. The serum used as a source of C' was diluted in HBS-BSA and was added at 50 µl/well. The plates were incubated at 37°C for 60 min and were spun down for 1 min at 3000 x g. Twenty-five microliters of lysate was carefully transferred to a replica plate, and hemolysis was estimated spectrophotometrically by registering the generation of fluorene blue at 630 nm in a ELISA reader as described (13). Each point was assayed in triplicate, and values obtained were expressed as percentage of lysis compared with a distilled water control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a first step in characterizing the recombinant proteins,
secretions from transfectants labeled by growth in the presence of
35S-methionine were immunoprecipitated with
antisera specific for H chain and were analyzed by SDS-PAGE without
reducing the disulfide bonds (Fig. 1
).
All transfectants secreted fully assembled
H2L2 molecules. IgG4 with
both and L chains also secreted HL half-molecules. The
additional bands migrating smaller than
H2L2 are not consistently
observed in the secretions and may represent some cell lysis during
labeling.
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). IgG1, IgG2, and IgG4 Abs with L
chains were rapidly assembled into
H2L2 molecules, with
H2L2 assembly virtually
complete by 15 min. In contrast, for IgG1, IgG2, and IgG4 30
min after the chase, the majority of the cytoplasmic Igs remained
as assembly intermediates. Both the - and -producing cell
lines had free L chain in the cytoplasm. For IgG1 and IgG2, the
predominant assembly intermediate was H2,
irrespective of whether the L chains were or . In IgG4, HL
appeared to be a more significant assembly intermediate; as noted
above, HL half-molecules are present in the secretions of
IgG4-producing cell lines. Both IgG3 and IgG3 assembled slowly,
and significant assembly intermediates remained 30 min after the chase.
Thus, these studies indicate that the isotype of both the H and L
chains can influence the assembly pathway and speed.
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When -chain-containing proteins were purified using
DNS-affinity chromatography, we found that assembly intermediates were
present (Fig. 3A). Although we
had routinely observed HL half-molecules in -chain-containing
proteins of the IgG4 isotypes, we had not observed intermediates in
assembly for purified proteins of the other isotypes. To obtain
homogenous proteins for functional studies,
H2L2 molecules were
isolated by size exclusion on Sepharose columns (Fig. 3B).
Reduction of the purified proteins yielded H and L chains of the
expected size (Fig. 3C).
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The isolation of incomplete Abs following affinity purification of
proteins suggested that free thiols present in the medium may be
exchanging with the disulfide bonds and breaking them. To more
completely investigate this possibility, purified
H2L2 molecules were treated
with varying concentrations of DTT and the cleavage of interchain
disulfide bonds were monitored by SDS-PAGE (Fig. 4). IgG1 was the most susceptible to
cleavage by DTT, with IgG1 somewhat more susceptible than IgG1.
At DTT:IgG disulfide ratios as low as 0.8, disulfide bonds are cleaved.
For IgG1, significant HL is seen, whereas for IgG1 the H-L bonds
appear to be broken before the H-H disulfide bond. The disulfide bonds
of IgG2, IgG3, and IgG4 are much more resistant to attack by DTT than
are those of IgG1. For IgG2, only at a DTT:IgG disulfide ratio of
100 are significant numbers of interchain disulfides broken; the
disulfides of IgG2 are more susceptible to attack than those of
IgG2 and, at this ratio, the Ab is almost completely reduced to H
and L chains. In contrast, IgG3 is more susceptible to DTT attack
than is IgG3, and at a DTT:IgG disulfide ratio of 100 it is
virtually completely reduced to H and L. At the same DTT concentration,
IgG3 shows only partial cleavage of its interchain disulfide bonds.
The disulfides of IgG4 are slightly more susceptible to cleavage
than are those of IgG4. It is noteworthy that for both IgG4s, the
H-H disulfide is broken before the H-L bond and significant amounts of
HL half-molecules are observed. Therefore, differences in
susceptibility to reducing agents were observed depending
on the isotypes of both the H and L chains. However, there was not a
consistent difference that depended only on the isotype of the L
chain.
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The constant region of the H chain is known to influence the in
vivo clearance of Ab molecules. To determine whether the isotype of the
L chain can also play a role, purified proteins were iodinated and
injected into the tail veins of BALB/c mice, and their elimination was
followed by whole-body counting over time (Fig. 5). For IgG1, IgG3, and IgG4, the isotype
of the L chain did not influence the kinetics of clearance, although
the isotype of the H chains did play a role, with IgG4 clearing most
rapidly and IgG1 clearing most slowly. However, for IgG2, the isotype
of the L chain contributed to the rate of clearance, with IgG2
clearing more rapidly than IgG2. Thus, for IgG2, but not for IgG1,
-3, and -4, the isotypes of the L chain appear to influence the
kinetics of elimination.
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When IgG1 and IgG3 at different concentrations were mixed with
DNS-coated SRBC in the presence of human complement, IgG3 was found to
be a more potent mediator of cytolysis than IgG1 (Fig. 6). However, the isotype of the L did not
influence the ability of the Abs to effect complement-mediated
cytolysis. IgG2 and IgG4 do not effectively activate complement and
were not tested.
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To determine the impact of the isotype of the H and L chain on the
kinetics of binding and affinity for Ag, proteins at varying
concentrations were added to DNS-BSA-coated IAsys biosensor cuvettes,
and the association and dissociation was followed with time (Fig. 7). The
ka,
kd, and
KD were then calculated (Table I). In general, IgG1, IgG2, and
IgG4 with L chains showed an increased
ka and increased
kd relative to the equivalent isotype
with L chains. The most profound difference was observed with IgG4,
and IgG4 showed a 2-fold increase in
KD compared with IgG4. This
increased KD was also evident in the
binding profiles with binding not observed for IgG4 at
concentrations less than 1 µg/ml. For all of these studies, proteins
were used that were H2L2
molecules, as indicated by SDS-PAGE and staining with Coomassie blue.
These studies indicate that the isotype of the L chain has at most a
slight impact on the equilibrium binding constants of the
anti-DNS Abs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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myeloma Wes showed that the interactions between the H
and L chains of F(ab')2 are extremely strong and
are maintained at guanidinium hydrochloride conditions sufficient to
denature the isolated domains. Although the C regions are primarily
responsible for maintaining the association, the V region also
contributes significantly to the association of the chains
when it is made unimolecular by its connection to the associated C
region (15). Examination of the in vitro reassembly of
F(ab')2 indicated that the high-affinity
association of Fd (VH CH1)
and a L chain derives from a combination of relatively weak
interactions involving pairs of domains
(VH-VL and
CH1-CL) (6).
Interestingly, the binding of one L chain domain to Fd induced a
conformation change in the adjacent domain, thereby modifying its
reactivity toward the complementary L chain domain.
During the in vivo assembly of the molecule, noncovalent association of
the subunits must precede interchain disulfide bond formation because
sulfhydryl groups must be brought close together before oxidation.
Recent studies have indicated that L chains must be assembled with
either a H chain or another L chain to be secreted (16).
Covalent assembly of H and L chains proceeds more slowly with L
chain and IgG1, IgG2, and IgG4 H chains, but not with IgG3 H chains. In
contrast, IgG3 is more completely assembled at 30 min than is
IgG3. IgG3 is distinguished by an extended hinge region that
provides extensive segmental flexibility, and this flexibility may make
it easier for the free disulfide of the L and IgG3 H chain to get
into close proximity and form a covalent bond. IgG1, IgG2, and IgG4 are
less flexible, and it would appear that the formation of the H-L
disulfide bond forms more slowly with than with L chains,
suggesting a different relative position of the disulfides with the
different L chains. Sequence comparison of and shows many
differences at positions predicted to form the
CH1-CL interface (Table II), and these may influence the
interactions between the domains during assembly.
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to be relatively
resistant to DTT-mediated cleavage, in contrast to what was observed
with thioredoxin, we found that the isotype of the L chain influences
DTT-mediated disulfide bond cleavage with IgG2 much more sensitive
to DTT-mediated reduction than IgG2.
It was surprising that changing the isotype of the L chain changed the
rate of in vivo clearance of IgG2. The neonatal FcR (FcRn) responsible
for transport of Ig across the neonatal rodent intestine has been
proposed to play a role in the control of the catabolism of IgG
(21, 22, 23, 24). FcRn binds to Fc at the interface between the
CH2 and CH3 domains
(25) with residues on the 252–257 and 307–311 loops in
CH2, and at positions 433–436 in
CH3 implicated in forming the recognition site
(reviewed in Refs. 26 and 27). Three
histidine residues present at the
CH2/CH3 interface are
responsible, at least in part, for the pH-dependent FcRn/IgG
interaction (22). Changing the isotype of the L chain
would not be expected to have any affect on the conformation of the
CH2/CH3 interface.
Therefore, it seems more likely that something other than the affinity
for FcRn is contributing to the differences in serum persistence seen
with IgG2 and IgG2. At the present time, it is not clear what
that is.
Human isotypes IgG1 and IgG3 are both effective in activating complement, IgG2 will activate only under selected experimental conditions, and IgG4 is inactive (28, 29, 30). The second constant domain (CH2) plays a critical role in complement activation and forms the binding site for C1q, although different residues have been found to play critical roles in Abs of differing isotypes (1, 31, 32, 33, 34). Because we observe no difference in the ability of IgG1 or IgG3 with L chains of different isotypes to activate complement, the L chain isotype does not appear to influence the conformation and accessibility of the C1q binding site within CH2 of either IgG1 or IgG3.
Using variable-region identical Abs, we have observed both L and H
chain-associated differences in binding kinetics. Similar
constant-region-related differences have been observed by others.
Differences in the association rate constants were seen with two
clonally related human monoclonal IgA1 and IgG1 Abs with
identical variable regions specific for tubulin (35). In
this study, the observed differences were seen with the
F(ab')2, suggesting that
CH1 played a role in structuring the Ag binding
site into a more kinetically competent form. In another study using
mouse-human chimeric anti-TAG72, the F(ab')2
from IgG1, -2, and -4 showed identical binding parameters
(36). However, different dissociation rate constants were
seen for the intact Igs with IgG4 < IgG3 < IgG2 < IgG1. In a study
using anti-4-hydroxy-3-nitrophenyl mouse-human chimeric Abs, the
same group again saw different kinetic parameters in Abs of different
isotypes. In these Abs, the L chain was murine (37).
For these Abs, ka was in the order
IgG4 > IgG2 > IgG3. IgG3 was found to have a 2-fold faster
association rate than IgG2 and IgG4; however, this increase in
association rate was countered by a >10-fold increase in dissociation
rate. In the present study, differences were seen in
ka and
kd depending on the isotype of the L
chains; however, Abs with the same H chains showed similar equilibrium
binding constants (Table I
). The greatest difference observed was the
slightly >2-fold differences in equilibrium binding constants seen
with IgG4.
It is not clear how the constant region structure of either the L or H
chain of the Ab is able to influence Ag binding characteristics. Using
murine variable-region identical Abs, IgG3 shows functional affinity
correlated with both slower dissociation rate constants and faster
association rates constants in comparison to other isotypes
(38). However, in the case of murine IgG3, this can be
explained by self-association between the constant-region domains
(39). Similar self-association has not been observed with
any of the Abs used in the present study. Segmental flexibility could
also contribute to the ability of the Ab to form multiple interactions
with Ag immobilized on a solid support. For the human constant
regions, IgG3 is the most flexible, followed by IgG1 and IgG4, with
IgG2 the least flexible (40). Thus, there is no obvious
correlation in the present studies between flexibility and the binding
parameters.
These studies have shown that the isotype of the L chain can influence
some of the properties of the Ab molecule, although it has a minimal
impact on most properties. The ability to produce two different L
chains arose early during the evolution of the immune system and has
been maintained. Different : ratios are seen in different
species; in this study, we have examined only human L chains, and we
may find different properties associated with the constant regions of L
chains from different species. In addition, we have analyzed only one
binding specificity, anti-DNS, a specificity initially associated
with a murine L chain. It will be of interest to expand these
studies to include additional binding specificities and Abs that
originally contained L chains of differing isotypes. We have also only
studied one of the functional constant regions present in humans.
It remains a puzzle as to why there are multiple, closely related,
constant regions in several species, including mouse and man.
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Footnotes |
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2 Current address: Instituto Venezolano de Investigaciones Cientificas, Centro de Medicina Experimental, Laboratorio de Patologia Celular y Molecular, Km. 11 Carretera Panamericana, Caracas, Venezuela.
3 Address correspondence and reprint requests to Dr. Sherie L. Morrison, Department of Microbiology, Immunology and Molecular Genetics, and Molecular Biology Institute, University of California, 405 Hilgard Avenue, Los Angeles, CA 90095-1489. E-mail address: sheriem{at}microbio.ucla.edu
4 Abbreviations used in this paper: anti-DNS, anti-dansyl; SH, sulfhydryl; FcRn, neonatal FcR.
Received for publication June 13, 2001. Accepted for publication October 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin-deficient mice. Eur. J. Immunol. 26:690.[Medline]
2-microglobulin-containing neonatal intestinal transport receptor. Proc. Natl. Acad. Sci. USA 93:5512.This article has been cited by other articles:
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  S. Das, N. Nikolaidis, J. Klein, and M. Nei Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers PNAS, October 28, 2008; 105(43): 16647 - 16652. [Abstract] [Full Text] [PDF]
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 J. Li, C. Zhang, T. Jostock, and S. Dubel Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site Protein Eng. Des. Sel., October 20, 2007; (2007) gzm038v1. [Abstract] [Full Text] [PDF]
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 J. Vera, B. Savoldo, S. Vigouroux, E. Biagi, M. Pule, C. Rossig, J. Wu, H. E. Heslop, C. M. Rooney, M. K. Brenner, et al. T lymphocytes redirected against the {kappa} light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells Blood, December 1, 2006; 108(12): 3890 - 3897. [Abstract] [Full Text] [PDF]
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