Mol. Cells, Vol. 2, pp. 115-118
Communication
Production of Nonagglutinating Monoclonal Antibodies against
Human Red Blood Cells
Suhn-Young 1m t and Young Ik. Lee*
Molecular Genetics Laboratory, Genetic Engineering Research Institute, Korea Institute of Science
and Technology, Tarjon 305-606, Korea
(Received on February 25, 1992)
Nonagglutinating monoclonal antibodies against human red blood cells were produced.
A volume of 2 X 107 Ficoll-paque purified red blood cells was used as antigen. Hybrid
clones which cause agglutination with red blood cells after adding anti-mouse IgG were
selected. Indirect agglutination tests were used for isotype determinations of these antibodies. Eighteen clones producing non-agglutinating antibodies against human red blood
cells have been isolated. Among them, sixteen clones produced IgG'(K), while the other
two clones produced IgG2b(K) antibodies. Ascites fluid from mouse injected with IgG'(K)
type hybridomas were purified, and heavy and light chains of immunoglobulin were identified to have molecular masses of 51 kDa and 25 kDa, respectively.
Important routes for transmission of the human immunodeficiency virus (HIV), hepatitis B virus (HBV)
and hepatitis C virus (HCV) -are transfusion and administration of product from contaminated blood
(Bianco et al., 1989, Imagawa et al., 1989). Thus, to
prevent transmission and wide spread of these viruses,
screening of donated blood is essential. For this purpose, enzyme linked immunosorbent assay (ELISA),
radioimmune assay (RIA) and Western blotting assay
have been used widely all over the world (Eva and
Perlmann, 1971; Sarngadharan et aI., 1984, Burke et
aI., 1987).
Although the assays are sensitive and useful for detection of antibodies against the viruses, many problems have been noticed (Blomberg and Klasse, 1988).
Usually it takes more than 3 h to determine the presence and absence of the virus antibodies from the
blood donors. Furthermore, extensive facilities such
as spectrophotometers (ELISA reader) and plate washers are needed. Evenmore, one has to handle the
blood in order to separate serum from the blood. It
could be dangerous, because one could be exposed
to dangerous viruses like HIV and HBV (Jennifer,
1988).
In order to overcome all these problems we generated nonagglutinating mouse monoclonal antibodies
against human red blood cells. By using these antibodies one can generate a rapid, and sensitive antibody
testing method using whole blood systems.
Materials and Methods
Immunization and generation oj mouse monoclonal
* To
whom correspondence should be addressed
t Present address: Presbyterian Medical Center, Christian
Medical Research Center, Chonju, Korea
antibodies
In order to collect human red blood cells, 10 ml
normal blood samples were used. Collected normal
blood samples were diluted two-fold with PBS. Ficollpaque (Pharmacia, Uppsala Sweden) gradients were
used to separate red blood cells from collected blood
samples (Parish et aI., 1974). Briefly, whole blood and
Ficoll-paque were prewarmed to 20 t in a water bath,
4 ml of Ficoll-paque were added to 10 ml polycarbonate tubes and 2-6 ml prewarmed whole blood were
layered on top of the Ficoll-paque layer. Tubes were
centrifuged for 20 min at 2,000 X g. After centrifugation, all the fluids were removed and the enriched
red blood cells were collected. The red blood cells
were washed three times with PBS. After washing, 2
X 107 red blood cells were used for intraperitoneal
injection into Balb/c mouse. The same number of cells were injected three times at three-week intervals.
Final injection was carried out with the same number
of red blood cells. After checking antibody titer using
hemagglutination test (Coombs, 1987), hybridization
with SP2/0 myeloma cells and screening of antibodyproducing cell lines were carried out using Kohler's
method (Kohler and Milstein, 1975).
Isotype determination
For selection of the clones which produce nonagglutinating antibodies from surviving clones in HAT
media, supernatants from these clones were tested. Supernatants which caused agglutinations directly were
eliminated, while those causing agglutination indirectly
were saved (Fig. 1). These clones produce antibodies
which cause agglutination when anti-mouse IgG were
added. This indirect agglutination test was used for
isotype determination of the antibodies. Anti-mouse
IgG" IgG2a, IgG 2b, IgG3, and IgM were used as heavy
chain determinants and anti-mouse K, and A as light
© 1992 The Korean Society of Molecular Biology
116
Mol. Cells
Nonagglutinating mAb against Human Red Blood Cells
agglutination
B. indirect
anti mouse
red cell
Figure 1. Basic schemes for direct and indirect agglutination reactions. The nonagglutinating monoclonal antibodies agglutinate
red blood cells using anti-mouse IgG as secondary antibodies.
chain determinants.
Purification of non agglutination monoclonal antibodies
In order to purifY nonagglutinating monoclonal antibodies against human red blood cells, ascites fluids
were generated using Balb/c mouse. After collecting
ascites fluids, IgG fractions were purified by the methods of Bruck et al. (1982), using OEAE-Affigel blue
chromatography: 1 ml of ascites fluid was centrifuged
at 15,000 rpm for 30 min at 40 °C, then dialysed against 20 roM Tris-HCl, pH 7.2, and layered on a OEAEAffigel blue chromatography. NaCI gradients of 0-100
roM were used as salt gradients. Purified antibodies
were confirmed using SOS-PAGE and immunodiffusion analysis.
Results and Discussion
In order to generate nonagglutinating monoclonal
antibodies against human red blood cells, Balb/c
mouse were immunized with 2 X 107 red blood cells
from normal healthy persons. After 2 injections with
red blood cells, mouse sera were collected in order
to check the antibody titers. Figure 2 shows the results
of direct hemagglutination reactions with mouse serum. Lane A shows that normal mouse serum does
not show hemagglutination reactions with red blood
cells, while lanes Band C show hemagglutination
reactions. Figure 1 shows the basic schemes of the
differences between direct and indirect agglutination
reactions. For the nonagglutinating monoclonal anti-
bodies, anti-mouse IgG were needed for the agglutination of red blood cells, while agglutinating monoclonal
antibodies did not need anti-mouse IgG. Hybridoma
clones which were sUIvived in HAT media and shows
agglutination reactions when we add anti-mouse IgG
were serially diluted in order to clone nonagglutinating monoclonal antibody producing cells. Eighteen
clones showed nonagglutinatig reaction with red blood
cells, while agglutination occurred when anti-mouse
IgG were added (Table 1).
Figure 3 shows the isotype determination of a monoclonal antibody against nonagglutinating red blood
cell surface antigens using hemagglutination test. Antimouse IgG J, IgG 2a, IgG 2b, IgG 3, and IgM were used
as secondary antibodies for subtype determination. For
light chain determination anti-mouse K and A chains
were used as secondary antibodies. Only the supernatants that show indirect agglutination reactions were
selected for isotype determinations. Lane 0 shows
Table 1. Isotype determination of 18 mouse monoclonal
antibodies obtained against nonagglutinating red blood cell
surface antigens
Isotype Direct
test
Indirect test
IgG' IgG. IgG2, IgG 2b IgG, IgM
+
+
a
+
Whole anti-mouse IgG
+
K
Number
of clones
A obtained
+ + -
16
2
Suhn-Young 1m & Young Ik Lee
Vol. 2 (1992)
117
A
B
c
Figure 2: Hemagglutinin titers against human red blood cells. Panel A shows the hemagglutinin test using normal mouse
serum. Panels B and C show the hemagglutinin test using immunized mouse serum. Balblc mice were immunized intraperitoneally using 2 X 107 human red blood cells on days 0 and 21 as described in Materials and Methods. Mice were bled
4 days after the last injection and were evaluated for anti-human red blood cell antibody titers employing direct hemagglutinin
assay. Sera were serially diluted for these tests.
\
D
2
ID
20SK - 116 99 - 66 ----45
3
4
5
6
7
8
9
~
h
~
I
--
29
19 M
k
N
Figure 3. Isotype determination of monoclonal antibodies
against nonagglutinating red blood cell surface antigens
using hemagglutination test. Anti-mouse IgG" IgG2a , IgG2b ,
IgG), and IgM were used as secondary antibodies for subtype determination. For light chain determination, antimouse and chains were used as secondary antibodies. Direct
(D) and indirect (ID) agglutination were determined as
shown in Figure 1.
Figure 4. SDS-polyacrylamide gel electrophoresis analysis of
monoclonal anti-human red blood cell antibodies after
DEAE-Affigel blue column elutions. Each peak fraction
from the column was run on 14% SDS-PAGE. Electrophoresis was carried out at room temperature for 22 h at 6
rnA, and the protein was visualized by coomassie blue staining. The heavy and light chains of immunoglobulins are
marked by h and I, respectively.
nonagglutination reactions of red blood cells and hybridoma supernatants, while lane ID shows agglutinations of red blood cells and hybridoma supernatant
by adding anti-mouse heavy and light subtype determinants. Among 18 clones, 16 clones have been identified to produce IgGJ(K), while the other 2 clones have
been identified IgG 2b (K) antibodies after isotyping analysis (Table 1). For the purification of nonaggiutinating antibodies, DEAE-Affigel blue columns were used
with 2 rnM Tris-HCl (PH 7.2) and 0-100 rnM NaCl
gradient as eluent. The eluted fractions were run on
SDS-PAGE in order to check the protein bands (Fig.
4). Lanes 4-6 show the fractions with distinct heavy
and light immunoglobulin chains, while the other lanes show albumin and other contaminating protein.
Immunoglobulin fractions (lanes 4-6) were pooled
and confirmed using Oucherlotony immunodiffusion
test. Goat anti-mouse IgG was used as a secondary
118
Nonagglutinating mAb against Human Red Blood Cells
Mol. Cells
antibody-peptide antigen causes agglutinating reactions
of red blood cells, while in the absence of secondary
antibodies (in normal persons' sera), antibody-antigen
complex does not cause any agglutinating reactions
of red blood cells.
Thus, the nonagglutinating antibodies can be used
for a simple and rapid diagnostic kit using whole
blood system. Using these systems one can overcome
time-consuming, expensive and sometimes dangerous
diagnostic steps in treating dangerous virus-contaminating bloods.
References
Bianco,
c.,
Uehlinger, J., and Kaplan, H. S., (1989)
New Engl. J Med. 321, 1678-1679
Figure 5. Oucherlotony immunodiffusion analysis of purified human red blood cell non-agglutinating monoclonal an tibodies against goat anti-mouse IgG. Center well contains
goat anti-mouse IgG. Well 1 contains a pool of proteins
from lanes 2 and 3 in Figure 4. Wells 2, 4, and 6 contain
pools of Ig peak proteins from lanes 4, 5, and 6 in Figure
4, respectively.
antibody. Our data shown in Figure 5 demonstrated
that non agglutinating monoclonal antibodies were
produced against human red blood cells. These antibodies can be used to generate a rapid and sensitive
diagnostic method using whole blood cells after conjugating with purified peptide antigens. Peptide antigens can be generated from any organisms of interest
using recombinant DNA technology or conventional
purification procedures. Under the presence of secondary antibodies (in patients' sera), nonagglutinating
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