Immunology Letters 86 (2003) 45 /51
www.elsevier.com/locate/Molecular characterization of a human monoclonal antibody to B
antigen in ABO blood type
Mika Kaneko, Yukinari Kato, Hidekazu Horiuchi, Motoki Osawa *Department of Forensic Medicine, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan
Received 26 August 2002; accepted 3 December 2002

Abstract
A human anti-B antibody of clone BT97 was obtained from a healthy individual of type A of the ABO blood group without
immunization. Cloning was performed by means of heterohybridoma formation of cell fusion between human peripheral
lymphocytes and mouse myeloma cells. The antibody selectively reacted with B-antigen in flow cytometry using red blood cells andenzyme-linked immunosorbent assay. The VH and VL genes of BT97 were derived from the germline genes of DP-47 and 3p.81A4,
respectively, with a couple of somatic mutational events. Comparative analysis with other reported anti-A, B and H antibodies
revealed that the amino acid sequence of the VH region was more homologous than that of the VL region. The sequence of BT97
showed complete identity with one anti-H natural antibody reported by Marks et al., with the exception of the CDR3 region. It is
not known whether the homologies include the common properties of the natural antibodies; however, a particular germline genepotentially changes to anti-ABH antibodies. We think that this method is suitable for cDNA preparation of human monoclonal
antibodies to blood group antigens and for sequence analysis.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Human monoclonal antibody; Heterohybridoma; Natural antibody; ABO histo-blood type

1. Introduction
Carbohydrate complexes combined with proteins andlipids decorate the plasma membrane of erythrocytes;
moreover, these species are often polymorphic, which is
known as blood types such as ABO, Ii and P type. In the
ABO blood group, ABH antigens, which are carbohydrate structures present in many tissues, are synthesizedunder the control of highly related genes coding for
glycosyltransferases [1]. The blood type A molecule isformed by an a1,3GalNAcT corresponding to the A
allele of the ABO locus. The blood type B allele encodes
an a1,3galactosyltransferase that forms the B determinate. O alleles encode functionally inert polypeptides

Abbreviations: mAb, monoclonal antibody; ELISA, enzyme-linked
immunosorbent assay; RBC, red blood cell; PBL, peripheral blood
lymphocyte; RT-PCR, reverse transcription-polymerase chain
reaction; CDR, complimentarity-determining region.
* Corresponding author. Tel.: /81-23-628-5271; fax: /81-23-6285273.
E-mail address: mosawa@med.id.yamagata-u.ac.jp (M. Osawa).

that do not further modify H-active precursors andtherefore represent null alleles at this locus. Four aminoacid substitutions were identified between A transferase
and B transferase. Most O alleles were found to containa single base deletion near the N-terminus of the coding
sequence. The phenotypes A, B, O and AB have beenclinically examined by hemagglutination for transfusion.
The antisera for laboratory examinations were, at one
time, derived from immunized individual donors; however, these antisera are no longer available as a result of
infectious hazard. To date, monoclonal antibodies(mAbs) to A and B-antigens raised in mice have
replaced antisera. mAbs are sufficiently effective forregular laboratory examinations; however, the mAbs areless valuable than the former human antisera in cases
involving the adsorption-elution test.
Antibodies to ABO antigen-structures are among themost predominant naturally occurring antibodies. TypeA individuals display anti-B antibodies in their plasma.
Type B individuals exhibit anti-A antibodies. Type O
individuals possess both antibodies. The antibodies areoligoclonal; additionally, they consist mainly of IgM [2].

0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0165-2478(02)00294-8

46

M. Kaneko et al. / Immunology Letters 86 (2003) 45 /51

Their high titer is maintained throughout the lifespanfollowing birth despite no apparent specific stimuli. The
physiological importance of natural antibodies remains
unclear; however, roles for natural antibodies including
nonspecific anti-bacterial defense and self-regulation of
the immune system have been proposed [3,4]. Despite
the classical theme, investigations concerning the production of specific antibodies by cloned cells display anumber of shortcomings, which may be attributable tothe difficulty associated with the production of humanmAbs.
In order to obtain a human immunoglobulin gene,two major technical approaches are widely applied. The
first technique involves the phage-display method, often
combined with lymphocyte-transformation by Epstein /
Barr virus (EBV) [5,6]. The second approach consists ofheterohybridoma formation between EBV-transformed
human lymphocytes and mouse myeloma cells [7 /9].
However, both methods possess critical limitations. In
the former technique, the combination of H and Lchains may or may not involve the original species
expressed in the specific antibody producing cells. In
contrast, production of human antibody is quite low
and rapidly diminished under mouse genomic control in
the latter case. However it is certain that the original
combination of H and L-chains is obtained by the fusion
protocol.
In the present study, in order to characterize thevariable region of anti-ABH antibody, the simplest
protocol of the fusion methodology between humanperipheral blood lymphocytes (PBL) and mouse myeloma cells was selected [10]. A clone producing anti-B
antibody was obtained. Following determination of thesequence coding the variable region of the specificantibody, a comparative analysis was conducted withthose anti-ABH antibodies previously reported.

2. Materials and methods
2.1. Cell fusion
Peripheral blood (100 ml) was extracted from ahealthy volunteer of type A. Mononuclear cells wereisolated by centrifugation on Ficoll-Paque Plus (Amersham Pharmacia Biotech); isolated cells were washedtwice with phosphate-buffered saline (PBS). Mouse
myeloma cells, P3U1, were cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum(FBS) (Morgate) at 37 8C in a saturated atmosphere of
5% CO2. Following harvest, myeloma cells were fusedwith human mononuclear cells of 1.2 /3.8 /108 at aratio of 1:10 in a warmed mixture of 1 ml of PEG-1000(Wako), 1 ml of FBS- free RPMI 1640 and 50 ml ofdimethylsulfoxide. After a 2-min incubation and centrifugation, the pellet was resuspended in 100 ml of

HAT selective medium (Gibco BRL) supplemented with
10% FBS in RPMI 1640 medium. To exclude fibroblast
cells, the cell suspension was placed on 75-cm2 culture
dishes overnight. The fused cells were gently collectedfrom the dishes, and subsequently transferred into 96well culture plates in a volume of 200 ml (2 /105 humancells per well). Following a 1-week incubation, half ofthe supernatant was discarded from each well and an
identical volume of HT medium was added. After 4
weeks, the supernatants were screened by flow cytometry analysis with red blood cells (RBCs) and enzymelinked immunosorbent assay (ELISA).
2.2. Flow cytometry analysis
RBCs of types A, B and O were prepared using
peripheral blood extracted from healthy volunteers. The
culture supernatant was incubated with 5 ml of the 3%
RBC suspensions for 1 h. After washing twice with PBS,
the RBCs were incubated with a 100-fold dilution of
FITC-conjugated goat anti-human IgM (m) (Kirkegaard& Perry Laboratories) for 1 h. The reactions were
performed at room temperature. Following additional
washing and fixation with 0.25% para-formaldehyde in
PBS, cells were analyzed with a FACS Calibur (Becton
Dickinson) flow cytometry device.
2.3. ELISA
A-and B-tri-oligosaccharides conjugated to humanserum albumin (IsoSep AB) were independently coatedonto microtiter plates (Sumitomo Bakelite) at 1 mg/ml
for 1 h. After blocking with 0.3% gelatin, the plates wereincubated with the culture supernatant for 1 h. Following washing with 0.05% Tween 20 in PBS, the plates
were incubated with rabbit antibodies to human IgM,
IgG, l light chain and k light chain (Dako) for 1 h,followed by peroxidase-conjugated goat F(ab?)2 to
rabbit IgG (Medical & Biological Laboratories) for 1
h. Following subsequent washing, the enzymatic reaction was conducted with a substrate solution consistingof 0.55 mg/ml 2,2?-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) and 0.03% H2O2. The optical density wasmeasured at 405 nm with an auto-plate reader. These
reactions were performed with a volume of 50 ml atroom temperature.
2.4. Isolation of the variable regions of immunoglobulin
cDNA
Total RNA was prepared from the heterohybridoma
cells employing a RNeasy mini kit (Qiagen). The initialstrand cDNA was synthesized by AVM-reverse transcriptase (Takara) via priming nine random oligomers
and oligo-dT. In order to amplify Fab portions, PCR
amplification was effected with oligonucleotide mixtures

M. Kaneko et al. / Immunology Letters 86 (2003) 45 /51

of VL/CL for k and l chains and of VH/CH1 for m andg chains. Primers were designed according to thosereported by Takekoshi et al. with minor modifications[11]. The reaction progressed with HotStar Taq polymerase (Qiagen). Reaction conditions were as follows:
the initial denaturation step occurred at 95 8C for 15
min, followed by 35 rounds of a temperature cycle at
95 8C for 30 s, 55 8C for 30 s and 72 8C for 1 min.
Following electrophoresis on 1% agarose gels, PCR
products were purified from the excised pieces utilizing aspin column (Gibco BRL). DNA fragments were subcloned into a plasmid vector, p123T (Mo Bi Tec);
subsequently, DNA fragments were labeled with
the BigDye terminator cycle sequencing kit and analyzed with the ABI prism 310 auto-sequencer (Perkin /
Elmer).
2.5. Sequence analysis
Nucleotide sequences of anti-ABH antibodies, retrieved from the DDBJ/EMBL/GenBank database,include the following accession numbers: ABO.B1VH /Z23026;
ABO.B1-VL /Z23027,
ABO.HI1VH /Z23028; ABO.HI1-VL /Z23029; HuA-VH /
L41175; HuA-VL /L41174. Nucleotide sequences of
A-fAb and B-fAb were provided by Dr Curiel-Quesada [12]. The sequence of FB5.7 was provided by Dr
Siegel [13]. To characterize the nucleotide sequence ofthe positive clones, a database search was conducted of
the IMGT (http://imgt.cines.fr:8104/home.html) [14], VBASE (http://www.mrc-cpe.cam.ac.uk/) and DDBJ/
EMBL/GenBank (http://www.ddbj.nig.ac.jp/) databases. Multiple alignments were effected employing the
CLUSTAL W program, ver. 1.60 [15]. Results were
illustrated with MACBOXSHADE, ver. 2.11. The aminoacid identity (%), designated to be the identicalamino acid number by the number of total regions
except for gaps, was calculated using the MACBOXSHADE
program.

47

ABH antigens. Hemagglutination was not readily apparent even in the presence of the Coombs reagent. As
displayed in Fig. 1, the presence of specific antibodies
was evident based on flow cytometry using RBCs. Whenthe supernatant from all wells containing colonies was
examined by flow cytometry analysis, three positive
clones were obtained, which secreted antibodies reactivewith B-RBCs. After a single step of limiting dilution,
one cloned hybridoma cell line was obtained, which was
designated BT97. The BT97 cells maintained antibody
production for approximately 10 weeks after fusion. To
ensure specificity and to determine the subclass, ELISA
was conducted employing soluble antigen coated plates(Fig. 2). BT97 mAb selectively reacted with the Bantigen as well. In addition, ELISA with subclassspecific second antibodies demonstrated that the BT97
mAb was comprised of Hm and Ll chains; moreover,
the whole serum of the donor also contained anti-B
antibodies composed of Hm and Ll chains.

3. Results
3.1. Cloning of human anti-B mAb
In order to obtain human antibodies to ABH antigens
mAb, heterohybridomas between human PBL andmouse myeloma cells, P3U1, were generated according
to the common procedure. Pre-culture in the dishes was
effective with respect to removal of excessive fibroblasts.
Hybridoma colonies were observed in 16/38% of theculture plates 4 weeks later. This observation indicatedthat the lymphocytes were successfully fused at afrequency of approximately 4 /10 7. Flow cytometry
analysis and ELISA were conducted to clone thehybridoma cells and to evaluate the antibodies toFig. 1. Flow cytometry analysis using B-RBC (upper) and A-RBC(lower) to detect positive hybridoma clones. The shadowed area
represents the peaks stained with the supernatant of BT97. Bold line
represents the peaks stained with human anti-A antiserum to B-RBC
and anti-B to A-RBC as a control. Thin line depicts the peaks stainedin the absence of the first antibody as a negative control.

48

M. Kaneko et al. / Immunology Letters 86 (2003) 45 /51

Fig. 2. Recognition of A and B antigens by the antibody of the BT97
clone supernatant and the donor antiserum. The microtiter plates were
coated with B-tri-oligosaccharides. The O.D. value is expressed as themean9/SD.

3.2. Sequence analysis of BT97 mAb
To deduce amino acid sequence of variable regions,reverse transcription-polymerase chain reaction (RTPCR) was performed on the RNA extract from theBT97 cells during production of the antibody. PCR
products were subcloned into a vector, followed bycomplete sequencing from both strands. The nucleotide
sequence of H and L-chain variable regions of the BT97
mAb are deposited in DDBJ/EMBL/GenBank databank (accession numbers AB087877 and AB087878,
respectively). The sequence of BT97 mAb was comparatively analyzed against several human mAbs to ABH
antigens that have been reported. B-fAb [12], ABO.B1[16] and FB5.7 [13] are anti-B antibodies, A-fAb [12]and HuA [17] are anti-A antibodies, and ABO.HI1 [16]is an anti-HI antibody. Five of the six reported antiABH antibodies were obtained employing the phagedisplay method. The remaining HuA was produced bycloned EBV-transformed lymphocytes that were stabilized by fusion with a human /mouse hybridoma [18].
In a fashion similar to BT97 mAb, five of the six antiABH antibodies, B-fAb, ABO.B1, A-fAb, HuA andABO.HI1, were comprised of Hm chains; the remaining
FB5.7 mAb demonstrated an Hg chain. The VH genesof BT97, B-fAb, ABO.B1, HuA, and ABO.HI1, belong
to IGHV3 in the IMGT classification, whereas the othertwo genes of FB5.7 and A-fAb belong to IGHV4. In
contrast, the VL and JL genes of these species exhibiteda variety of the IGLV and IGLJ groups.
Based on their homology, it was evident that the VH
and VL genes of BT97 were derived from the germline
DP-47 gene [19] corresponding to the IGHV3 subgroupin the IMGT database, and the germline 3p.81A4 [20]corresponding to the IGLV3-10 subgroup, respectively.
ABO.B1 and ABO.HI1 were also derived from the DP47 gene. B-fAb and HuA were derived from the DP-46
gene. Additionally, FB5.7 and A-fAb were derived
from the DP-70 and DP-67 genes, respectively. Fig. 3
shows the multiple alignment along the VH and VL
regions among the eight anti-ABH mAbs including
BT97 and its germlines. It was clear that the VH genes
revealed high homology, with the exception of the third
complimentarity-determining region (CDR3). In contrast, the VL genes displayed less homology than that of
the VH genes. Employing this multiple alignment, theamino acid sequence identity (%) of the CDR and theframework (FR) regions in the VH and VL genes ofBT97 to the germline and the six antibodies wascalculated (Table 1). Germline sequences (DP-47 and3p.81A4) did not completely include the CDR3 region
and its downstream regions; thus, the estimation of theidentity of the CDR3 region was relatively low. The VH
gene of BT97 and DP-47 are identical, with the
exception of a three-nucleotide difference in the regionof the 5? primer used in PCR. Thus, the differences
between the VH gene of BT97 and DP-47 in the FR1
region had potentially been introduced by mismatches
during the PCR priming cycles. The VL gene of BT97
differs from 3p.81A4 by seven nucleotides, which areaccompanied by two amino acid substitutions in the
CDR3 region. Except for these differences, the VH and
VL genes of BT97 exhibited no other substitution in
their nucleotide sequences. With respect to the FR
regions of the VH gene of anti-ABH antibodies, highhomology was evident with 53 /100% residual identities.
Particularly, BT97 and ABO.HI1 possessed identicalsequences in the VH gene except for the CDR3 region
downward. On the other hand, FR regions of the VL
gene displayed identities of 35/78%.

4. Discussion
We obtained a human mAb of BT97 that reacted with
B-antigen of the ABO blood group. Heterohybridoma
formation in this study is characterized by methodological limitations, including low fusion rate and unstableantibody production. Large volumes of the antibodycontaining supernatant were not obtained; however, it
was sufficiently effective to clone the cells and to prepare
cDNA coding the specific antibody. Although thisprotocol is in need of improvement, the advantagethat the combination of the obtained H and L-chainsis original indicates that this approach is suitable for the
analysis of the sequence of immunoglobulins.
As screening procedures, hemagglutination and
ELISA are common in preparation of anti-RBC antibodies [12,13,16,17]. In this series of experiments,
hemagglutination was rarely observed with the culture

M. Kaneko et al. / Immunology Letters 86 (2003) 45 /51

49

Fig. 3. The multiple alignment of the amino acid sequence of the H and L-chains. Amino acids are designated by single-letter code. Amino acids
identical to BT97 are shown on a black background; amino acids similar to BT97 are highlighted with a gray background. The CDR and FR regions
defined by the IMGT database are indicated over the top; those defined by the V-BASE are in parenthesis at the bottom.

[16]
18
78
33
71
33
96

100

100

100

100

23

66
87
Anti-HI
ABO.HI1

72

[12]
[17]
27
18
64
64
0
33
59
71
11
14
13
20
58
92
50
13
59
71
44
75
60
80

100
76
53
38
63
25
82
88
71
100
63
0
88
92
60

56
51
57
67
Anti-A
A-fAb
HuA

48
39

[12]
[16]
[13]
18
18
18
69
61
56
33
33
33
59
53
59
0
14
11
56
35
44
57
46
50
89
79
52
Anti-B
B-fAb
ABO.B1
FB5.7

100
100
100
100
88

13
7
19

98
97
Germ line
DP-47
3p.81A4

FR3
CDR2
FR2
CDR1
FR1

13

Total length
Total length
Antibody

CDR3

VL region
VH region

Table 1
Amino acid identity (%) of VH and VL regions compared to BT97 mAb

100

FR1

100

CDR1

100

FR2

100

CDR2

100

FR3

64

CDR3

[19]
[20]

M. Kaneko et al. / Immunology Letters 86 (2003) 45 /51

Reference

50

supernatant, which may be attributable to a low
concentration of the antibody. In addition, ELISA,
utilizing plates coated by the artificially synthesizedsoluble antigens, was not able to determine whether theobtained values precisely reflected the actual interactionof the antibody to RBCs. For these reasons, analysis byflow cytometry has been established in our trial. Flowcytometry employing RBCs was more sensitive anddemonstrative than was hemagglutination.
The nucleotide sequence of the variable regions of theBT97 mAb was determined by RT-PCR; moreover, theH and L-chains were derived from the DP-47 and3p.81A4 germline genes, respectively. Both the VH
and VL genes shared high amino acid identities of 97
and 98%, respectively, with their corresponding germline genes. These genes appeared to contain a minimum
number of somatic mutational events, suggesting that agermline gene such as DP-47 might originally have anaffinity to ABH antigens. It has been repeatedly
suggested that fetal antibodies and auto-antibodies are
dominated by rarely mutated or unmutated germline
VH genes and that these antibodies are often polyreactive [21 /24]. We think that the antibodies to ABHantigens appear to be naturally occurring in therepertoire of germline genes.
In an attempt to evaluate the amino acid sequenceand its organization of the BT97 mAb, a comparative
analysis was performed with the six reported anti-ABH
antibodies. Among these anti-ABH antibodies, theamino acid sequence of the H-chains exhibited greater
homology than that displayed by the L-chains. The
human mAbs of B-fAb, FB5.7, A-fAb and HuA wereobtained from immune disease patients. ABO.B1 andABO.HI1 were acquired from a normal human; however, Siegel et al. notes that the donor for ABO.B1 was
immunized [25]. Therefore, only two mAbs of BT97 and
ABO.HI1 are naturally occurring mAbs. Of particularinterest, the VH region of BT97 showed the highest
homology with ABO.HI1, with the exception of the
CDR3 region. As for natural anti-ABH antibodies, thereactive species are possibly derived from the samegermline genes. Furthermore, since BT97 is anti-B and
ABO.HI1 is anti-HI, the differences in the CDR3 of the
VH region or the VL region may distinguish the antigen
structures. Anti-B mAb of B-fAb also exhibited highhomology to BT97 with the complete identities in the
CDR1 and FR3 regions of the VH gene. In contrast,
FB5.7 revealed the lowest homology in the VH region
with BT97, particularly of the CDRs, which should becaused by the step of affinity maturation.
In conclusion, we obtained human anti-B mAb,
BT97, via human /mouse heterohybridoma formation.
Comparative sequence analysis of variable regions was
performed with previously reported species. Strong
homology, particularly in CDRs of the H chain, wasrevealed. The variety of antibody repertoire indicates theM. Kaneko et al. / Immunology Letters 86 (2003) 45 /51

general occurrence of natural antibodies in the ABO
blood group, whereas the ternary structure of thespecific antibodies should be constrained for the carbohydrates of antigen. In the future, further accumulationof data regarding the sequence of specific antibody
repertoire will be necessary in order to draw a conclusion. Experiments involving targeted mutagenesis arealso required to identify the antigen-reactive site on
antibodies.

Acknowledgements
We thank Dr E. Curiel-Quesada, Departamento de
Bioquimica, Escuela Nacional de Ciencias Biologicas delI. P. N., and Dr D.L. Siegel, Department of Laboratory
Medicine and Pathology, University of Pennsylvania,
who kindly provided the sequences of their anti-ABH
antibodies. This work was supported by a Grant-in-Aid
for Scientific Research, 13770218, from the Ministry of
Education, Culture, Sports, Science and Technology ofJapan.

References[1] F. Yamamoto, H. Clausen, T. White, J. Marken, S. Hakomori,Nature 345 (1990) 229 /233.
[2] R. Rieben, A. Frauenfelder, U.E. Nydegger, Eur. J. Immunol. 22(1992) 2129 /2133.
[3] P. Casali, A.L. Notkins, Immunol. Today 10 (1989) 364 /368.
[4] S. Avrameas, Immunol. Today 12 (1991) 154 /159.
[5] K. Thompson, G. Barden, J. Sutherland, I. Beldon, M. Melamed,
Immunology 71 (1990) 323 /327.
[6] M. Takekoshi, F. Maeda, Y. Nagatsuka, S. Aotsuka, Y. Ono, S.
Ihara, J. Biochem. (Tokyo) 130 (2001) 299 /303.
[7] R. Eren, I. Lubin, D. Terkieltaub, O. Ben-Moshe, A. Zauberman,
R. Uhlmann, T. Tzahor, S. Moss, E. Ilan, D. Shouval, E. Galun,

51

N. Daudi, H. Marcus, Y. Reisner, S. Dagan, Immunology 93(1998) 154 /161.
[8] M. Numasaki, K. Nakamura, Y. Fukuoka, N. Sato, H. Saeki, T.
Tachibana, N. Hanai, T. Nukiwa, T. Kudo, Immunol. Lett. 60(1998) 111 /120.
[9] T. Kudo, H. Saeki, T. Tachibana, J. Immunol. Methods 145(1991) 119 /125.
[10] C.F. Jessup, H. Baxendale, D. Goldblatt, H. Zola, J. Immunol.
Methods 246 (2000) 187 /202.
[11] M. Takekoshi, F. Maeda, H. Tachibana, H. Inoko, S. Kato, I.
Takakura, T. Kenjyo, S. Hiraga, Y. Ogawa, T. Horiki, S. Ihara, J.
Virol. Methods 74 (1998) 89 /98.
[12] E. Santos-Esteban, E. Curiel-Quesada, Vox. Sang. 81 (2001) 194 /
198.
[13] T.Y. Chang, D.L. Siegel, Transfusion 41 (2001) 6 /12.
[14] M. Ruiz, V. Giudicelli, C. Ginestoux, P. Stoehr, J. Robinson, J.
Bodmer, S.G. Marsh, R. Bontrop, M. Lemaitre, G. Lefranc, D.
Chaume, M.P. Lefranc, Nucleic Acids Res. 28 (2000) 219 /221.
[15] J.D. Thompson, D.G. Higgins, T.J. Gibson, Nucleic Acids Res.
22 (1994) 4673 /4680.
[16] J.D. Marks, W.H. Ouwehand, J.M. Bye, R. Finnern, B.D.
Gorick, D. Voak, S.J. Thorpe, N.C. Hughes-Jones, G. Winter,
Biotechnology (NY) 11 (1993) 1145 /1149.
[17] K.G. Nickerson, M.H. Tao, H.T. Chen, J. Larrick, E.A. Kabat, J.
Biol. Chem. 270 (1995) 12457 /12465.
[18] S.K. Foung, S. Perkins, A. Raubitschek, J. Larrick, G. Lizak, D.
Fishwild, E.G. Engleman, F.C. Grumet, J. Immunol. Methods 70(1984) 83 /90.
[19] I.M. Tomlinson, G. Walter, J.D. Marks, M.B. Llewelyn, G.
Winter, J. Mol. Biol. 227 (1992) 776 /798.
[20] S.C. Williams, J.P. Frippiat, I.M. Tomlinson, O. Ignatovich, M.P.
Lefranc, G. Winter, J. Mol. Biol. 264 (1996) 220 /232.
[21] P.P. Chen, N.J. Olsen, P.M. Yang, R.W. Soto-Gil, T. Olee, K.A.
Siminovitch, D.A. Carson, Int. Rev. Immunol. 5 (1990) 239 /251.
[22] J.L. Hillson, R.M. Perlmutter, Int. Rev. Immunol. 5 (1990) 215 /229.
[23] K.A. Siminovitch, P.P. Chen, Int. Rev. Immunol. 5 (1990) 265 /
277.
[24] V. Pascual, J.D. Capra, Adv. Immunol. 49 (1991) 1 /74.
[25] D.L. Siegel, M. Czerwinski, S.L. Spitalnik, Transfus. Clin. Biol. 9(2002) 83 /97.