The thymidine kinase-lacking hybridoma of the invention has resistance to oaubain and an Ig-producing ability and is formed by fusing a chicken B lymphoblast cell with an immunized chicken spleen cell. The hybridoma can be used as parental cell line for cell fusion, and the fused cell is excellent in the production of IgG.

BACKGROUND OF THE INVENTION 
This invention relates to an established hybrid cell having an 
IgG-producing ability obtained from a chicken, its preparation method, and 
a method of producing an antibody utilizing this hybrid cell. 
It is known that chicken-immunized globulin IgG has a very low 
cross-reactivity with IgG derived from a mammal (Hadge, D., et al, Mol. 
Immunol., 21, 699-707, 1984). Moreover, it is also known that the chicken 
IgG does not bind protein A (Guss, B. et al, EMBO J., 5, 1567-1575, 1986). 
Furthermore, the chicken antibodies have the advantages of not activating 
the complement system and not reacting with the rheumatoid factor in 
mammalian sera (Larsson, A., et al, I. Immunol. Methods, 108, 205-208, 
1988). Thereupon, an assay for measuring circulating immune complexes 
using a chicken anti-human complement antibody has recently been 
established (Largson, A., et al, J. Immunol. Methods, 113, 93-99, 1988). 
These facts indicate that chicken antibody is extremely useful in 
mammalian immunology field. Therefore, it is considered that, if a chicken 
monoclonal antibody can be supplied, the antibody can be utilized as a 
useful means not only in the field of avian immunology but also in that of 
mammalian immunology. 
The present inventors eagerly investigated in order to establish a parental 
cell line for the preparation of a chicken monoclonal antibody, and 
examined to produce a cell line lacking an enzyme necessary for selection 
of hybridomas from chicken B cells, similar to a mouse myeloma cell. As a 
result, we obtained a cell which can grow stably among 
thioguanine-resistant cells. However, all of the thioguanine-resistant 
cells have HAT (hypoxanthine-aminopterin-thymidine) resistance. Thereupon, 
we further investigated, and as a result, we established a thymidine 
kinase-lacking cell line with HAT sensitivity (HU3R cell line) which can 
stably multiply. Thus, we found that, when the established cell was fused 
with an immunized chicken spleen cell, a chicken monoclonal antibody could 
be accumulated in a culture medium by culturing the fused cell (the 
specification of Japanese patent KOKAI No. 2-186980, Int. Arch. Allergy 
Appl. Immunol., 88, 416-419 (1989)). 
However, antibodies produced by the hybridoma formed by fusing the HU3R 
cells are not IgG but IgM, and moreover, the cells lost the 
antibody-producing ability rapidly during sub culturing. 
Then, we further investigated, and obtained thymidine kinase-lacking cells 
(R27H cell line) from the above hybridoma. The thymidine kinase-lacking 
cells were fused with chicken spleen cells immunized with an antigen to 
produce novel hybridomas (J. Immunol. Methods, 139, 217-222, 1991, U.S. 
Pat. No. 5,411,881). As a result, it became possible to obtain hybridomas 
which could produce a specific antibody stably, and several chicken 
monoclonal antibodies were produced by using the hybridomas and 
applications thereof were reported (Animal Cell Technology: Basic & 
Applied Aspects, 527-534, 1992, Immunology Letters, 32, 91-96, 1992). 
However, multiplication of cells, which had been transformed by chicken 
retroviruses carried by the parental chicken B cells, frequently occurred 
during fusions with the R27H cells and were capable of growing in a in HAT 
medium which resulted in the difficulty of the multiplication of only 
hybridomas formed through the fusion selectively. 
That is, the cells transformed by retroviruses can multiply in a HAT 
medium, and the growth rate of the cells is greater than the hybridomas. 
The appearance frequency of the cells is greater than the hybridomas in a 
culture after cell fusion. The cells secreted antibodies even after a 
short period. Since the multiplication of the transformed cells interferes 
with the multiplication of newly formed hybridomas, it is necessary to 
develop a countermeasure therefor. 
SUMMARY OF THE INVENTION 
An object of the invention is to provide a chicken cell capable of being 
used as a parental cell for production of a chicken monoclonal IgG 
antibody wherein hybridomas formed by fusing the cell multiply in 
precedent to others, especially the transformed cells. 
The inventors investigated eagerly in order to achieve the above object, 
and have succeeded in obtaining a cell line which has achieved the object 
from the R27H cells by imparting a resistance to oaubain which is a 
specific inhibitor to Na.sup.+, K.sup.+ --ATPase. 
Thus, the present invention provides a thymidine kinase-lacking hybridoma 
having a resistance to ouabain and an Ig-producing ability in which a 
chicken B lymphoblast cell is fused with an immunized chicken spleen cell.

DETAILED DESCRIPTION OF THE INVENTION 
The cells of the invention can be obtained by culturing a thymidine 
kinase-lacking hybridoma having an Ig-producing ability and formed by 
fusing a chicken B lymphoblast cell with an immunized chicken spleen cell, 
in a culture medium containing ouabain, and isolating a hybridoma having 
resistance to ouabain. 
The established thymidine kinase-lacking hybrid cell is obtained from 
chicken B lymphoblast. The kind of chicken is not limited, and for 
example, white leghorn, white rock and the like can be utilized. 
The chicken B lymphoblast cell line is obtained from a chicken B 
lymphoblast cell suffering from cancer by a chicken retrovirus. 
The B lymphoblast cell line having self proliferation potency thus obtained 
is mutated, and thymidine kinase-lacking cells are selected. The mutation 
may be conducted by a physical means, such as UV irradiation, or by 
utilizing an agent, such as ethyl methanesulfonic acid (EMS), 
nitroguanidine or ICR-191. The isolation of the thymidine kinase-lacking 
cell from the mutant cells may be conducted, for example, by culturing in 
a culture medium containing a thymidine analog, such as trifluorothymidine 
(TFT) or bromodeoxyuridine, and cloning. The culture medium may be a 
conventional medium for cell cultures, for example, RPMI 1640 medium, 
Dulbecco's modified MEM medium or the like to which 5-15% of fetal bovine 
serum (FBS) or the like is added. The culture conditions may also be 
similar to conventional cell culture conditions and, for example, may be 
at about 37.degree.-41.degree. C. in an atmosphere of air to which about 
5-10% of CO.sub.2 is added. 
The established thymidine kinase-lacking cell thus obtained has a self 
proliferation potency, but it dies when cultured in a HAT medium. Besides, 
when it is measured by the indirect fluorescent antibody method, no cell 
synthesizes chicken Ig. This cell is designated as the HU3R cell. 
The HU3R cell is fused with an immunized chicken spleen cell . The spleen 
cell may be prepared by injecting several times an antigen, such as 
inactivated Newcastle disease virus, together with an adjuvant, into a 
chicken, and excising it after breeding. The fusion is conducted by a 
known cell fusion technique, such as polyethylene glycol, electric fusion 
or PVJ virus. 
The hybrid cell obtained is further mutated, and a thymidine kinase-lacking 
cell line is selected. It is preferable that these treatments are 
conducted after the subculture of the hybrid cell is continued until the 
growth becomes stable. The mutation treatment and the selection of 
thymidine kinase-lacking cells may be conducted similar to the 
aforementioned method. Besides, if necessary, mutation means and/or 
conditions may be changed. The production of Ig can be detected by 
detecting--.gamma. chains, .mu. chains and L chains which are a part of 
Ig, and the detection of these chains may be conducted according to a 
known method. For example, the indirect fluorescent antibody method using 
an anti-chicken Ig anti-.gamma. chain antibody and a fluorescein-labeled 
second antibody, flow cytometry, etc. can be utilized. The production of 
Ig can be confirmed by the confirmation of one of a .gamma. chain, .mu. 
chain or L chain. 
The thymidine kinase-lacking cell has HAT sensitivity and Ig-producing 
ability. Examples of the cell are R27H1 (FERM BP-3475), R27H4 (FERM 
BP-3478), etc. 
Subsequently, ouabain resistance is imparted to the cell. A means for 
imparting the ouabain resistance is to mutate the cell by a physical 
means, such as UV irradiation, or by using an agent, such as EMS, 
nitroguanidine or ICR-191, followed by cloning the mutant cells by 
culturing them in a medium containing ouabain. The culture medium may be a 
conventional medium for cell culture, for example, RPMI 1640 medium, 
Dulbecco's modified MEM medium or the like to which 5-15% of fetal bovine 
serum (FBS) or the like is added. It is preferable that the mutant cell is 
first cultured at an ouabain concentration of about 1.times.10.sup.-7 
-1.times.10.sup.-5 M, and the ouabain concentration elevated according to 
the multiplication of the cell. A suitable final concentration is about 
5.times.10.sup.-5 -5.times.10.sup.-4 M. The culture conditions may also be 
similar to those of conventional cell culturing, and, for example, may be 
at about 37.degree.-41.degree. C. in an atmosphere of air to which about 
5-10% of CO.sub.2 is added. 
The ouabain-resistant cell is cultured in an HAT medium to confirm that all 
cells died completely after 5-6 days from the start of culturing. The 
expression of Ig can be confirmed by the indirect fluorescent antibody 
method using anti-chicken Ig antibody and fluorescein-labeled secondary 
antibody, or the like. 
The established hybrid cell having Ig-producing ability thus obtained is 
further fused with an immunized chicken spleen cell. The immunization and 
the fusion may be similar to the aforementioned method. Since the antigen 
used for immunization is considered to impart IgG-producing ability to the 
cell after fusion with the immunized chicken spleen cell, the antigen is 
selected according to the desired IgG. 
The IgG may be produced by culturing the hybrid cell similarly to the 
aforementioned method, and the IgG can be produced and accumulated in the 
culture supernatant by culturing for about 1-30 days. Separation of IgG 
may be conducted by utilizing a known means, such as affinity 
chromatography, gel filtration, ion exchange chromatography, ethanol 
fractionation, rivanol fractionation, PEG fractionation, etc. 
According to the invention, specific antibody-producing cells can be 
obtained in a high efficiency by fusing a thymidine kinase-lacking 
ouabain-resistant cell line, e.g. MuH1 or MuH4, as a parental cell line, 
and growth of the specific antibody-producing cells is stable. Growth of 
retrovirus-transformed cells has not been found at all. The production of 
antibody is stable, and chicken IgG can be produced in quantity. The 
antibodies produced are monoclonal, and can be utilized not only in 
fundamental studies in chicken immunity but also as antibodies for 
medicines and clinical assay reagents. 
EXAMPLE 
I. Preparation of Ouabain-Resistant HAT-Sensitive Cell Line 
I-1. Chicken Cell Line 
R27H1 (FERM BP-3475) and R27H4 (FERM BP-3478) used in this example are 
parental cell lines established in previous studies. R27H1 and R27H4 were 
cultured in Iscove's modified Dulbecco's medium (IMDM) containing 10 
.mu.g/ml TFT and 10% FBS, placed in an incubator at 38.5.degree. C. in an 
air atmosphere containing 5% CO.sub.2, and subcultured for 2-4 days. 
I-2. Preparation of Ouabain-Resistant cell 
72.88 mg ouabain was dissolved in a phosphate buffer solution (PBS(-)) so 
as to become 20 ml (5.times.10.sup.-3 M) and sterilized by a 0.45 .mu.m 
sterilization filter. Then, the ouabain solution was put into 
sterilization tubes, and stored at 4.degree. C. under dark conditions. 
Mutation of R27H1 and R27H4 was carried out by suspending R27H1 cells or 
R27H4 cells in a RPMI 1640 medium containing 5% FBS in a concentration of 
2.times.10.sup.5 cells/ml, culturing at 38.5.degree. C. in a 5% CO.sub.2 
incubator for 6 hours, adding EMS to the medium at a concentration of 600 
.mu.g/ml, and further culturing for 24 hours. After culturing, cells were 
washed three times with RPMI 1640 medium to remove EMS, and then further 
cultured by suspending them in a RPMI 1640 medium containing 10% FBS for 
the purpose of the expression of mutation. 
After the mutated cells appeared from culturing 2-4 days, the cells were 
suspended in an IMDM medium containing 1.times.10.sup.-5 M ouabain and 10% 
FBS at a concentration of 1.times.10.sup.-5 cells/ml, and 100 .mu.l of 
cell suspension was put into a 96 well plate for tissue culture. After 
culturing at 38.5.degree. C. in a 5% CO.sub.2 incubator for several days, 
the grown cells were further allowed to grow in the same medium as 
mentioned above. When the multiplication became stable, the reagent 
concentration of the medium was gradually elevated to 1.times.10.sup.-4 M. 
I-3. Cloning of Ouabain-Resistant Cells by Soft Agar Method 
Cloning of the ouabain-resistant cells thus obtained was conducted by 
culturing on a soft agar medium containing 1.times.10.sup.4 M ouabain. 
After about two weeks culturing, colonies that grew very well on the soft 
agar were taken out and cultured in a growth medium containing 
1.times.10.sup.-4 M ouabain, at 38.5.degree. C. in a 5% CO.sub.2 
incubator, and particularly well grown clones were selected from the 
obtained clones. When the selected clones were cultured in a HAT medium 
containing 1.times.10.sup.-4 M hypoxanthine, 0.8.times.10.sup.-7 M 
aminopterin and, 1.6.times.10.sup.-5 M thymidine, all the cells were dead 
completely dead 5-6 days after the start of culturing. Accordingly, these 
clones could be used as a parental cell for cell fusion, and were 
designated as MuH1 (FERM BP-5442) and MuH4 (FERM BP-5443). 
II. Properties of Ouabain-Resistant HAT-Sensitive Cell Lines 
II-1. Sensitivity of Cell Lines Obtained by Cloning to HAT Medium 
Sensitivity of the cell lines obtained by the cloning to HAT medium was 
examined. Cells to be tested were suspended in a RPMI 1640 medium 
containing 10% FBS at a concentration of 1.times.10.sup.5 cells/ml, and 
100 .mu.l of cell suspension was put into each well of a 96 well plate for 
tissue culture. 100 .mu.l of a RPMI1640 medium containing 10% FBS, 
1.times.10.sup.-4 M hypoxantine, 1.6.times.10.sup.-5 thymidine and 
0.05-2.times.10.sup.-7 M aminopterin was added to each well, and cultured 
at 38.5.degree. C. in a 5% CO.sub.2 incubator. Cells in three wells of 96 
well plate were recovered at the start, after 2, 4, 6, 8, 10 and 14 days, 
respectively, and a dead cell rate was determined. 
II-2. Detection of Cytoplasmic Immunoglobulin (cIg) Expressing in 
Ouabain-Resistant Cell Line 
Expression of cIg in ouabain-resistant cell lines was examined by the 
indirect immunofluorescent method. Cells to be tested were transferred to 
a tube and washed three times with cold PBS (-). A small amount of a cell 
suspension in a high concentration was placed on a cover glass previously 
washed with ethanol and applied uniformly by a Pasteur pipette. After 
air-drying, the cover glass was put in a test tube, and a sufficient 
amount of an acid alcohol (acetic acid: ethanol=5:95) which was previously 
chilled to -20.degree. C. was put into the test tube to fix the cells by 
leaving at -20.degree. C. for 15 minutes. After air-drying, a suitable 
amount of rabbit anti-chicken L chain, rabbit anti-chicken .mu. chain and 
rabbit anti-chicken .gamma. chain diluted 50 times, 80 times and 60 times, 
respectively was placed on the cover glass as primary antibodies, and 
allowed to react at 37.degree. C. for 2 hours or at 4.degree. C. overnight 
in a moistened box. After the reaction was completed, the cells were 
washed 5 times with cold PBS (-). Fluorescein-labeled goat anti-rabbit IgG 
diluted 40 times was put as a secondary antibody, and allowed to react at 
37.degree. C. in a moistened box. After 1 hour, the cells were 
sufficiently washed, and then sealed by placing a solution prepared by 
mixing non-fluorescent glycerin and PBS (-) at a mixing ratio of 9:1 on 
the cover glass, and observed by a fluorescence microscope. 
II-3. Measurement of Cell Growth Rate 
The generation time of ouabain-resistant cells was measured. Cells to be 
tested were suspended in a RPMI 1640 medium containing 10% FBS at a 
concentration of 2.times.10.sup.5 cell/ml. 1 ml of the cell suspension was 
put into each well of a 24 well plate for tissue culture, and cultured at 
38.5.degree. C. in a 5% CO.sub.2 incubator. Cells from three wells were 
recovered every day and the number of living cells and dead cells were 
counted. Respective numeral values were plotted on a semilogarithmic paper 
from the start of culture to 1 week thereafter to prepare a growth curve. 
The generation time was calculated by the following formula. 
Generation time; g is, 
EQU g=(t.sub.2 -t.sub.1)13.32.times.log (x.sub.2 /x.sub.1) 
t.sub.1, t.sub.2 : time (hours) 
x.sub.1, x.sub.2 : number of living cells 
II-4. Detection of Chicken Immunoglobulin 
Chicken immunoglobulin in culture supernatants of ouabain-resistant cells 
was detected by the western blotting method. Ouabain-resistant cells to be 
tested were cultured in a serum-free IMDM medium for 24 hours, and the 
culture supernatant was added to 50% ammonium sulfate. Precipitates were 
collected and mixed with an electrophoresis buffer containing 
2-mercaptoethanol (2-ME). The mixture was boiled at 100.degree. C. for 3 
minutes and used as the sample for electrophoresis. SDS polyacrylamide gel 
electrophoresis (SDS-PAGE) was conducted using 10% acrylamide gel. 3-5 
.mu.g of a sample was charged into each lane of a minislab gel, and 
allowed to migrate at 20-40 mA. 
Thereafter, the migrants were transferred to a nitrocellulose membrane by 
the semidrying method, and color bands developed using antibodies were 
observed. 
The antibody to chicken immunoglobulin used as the primary antibody above 
was prepared as follows. 250 .mu.l of a PBS (-) solution. containing 100 
.mu.g chicken IgG or chicken IgM was mixed with an equal volume of FCA. A 
female Balb/c mouse was immunized by peritoneal injection, and then, 
further immunized with 125 .mu.l of chicken IgM or IgG solution 
intravenously as a secondary immunization. Thereafter, the spleen cell was 
fused with mouse myeloma cell SP 2/0-Ag14 by a known cell fusion method, 
and the antibody was obtained by culturing the hybridoma. Thus, 
anti-chicken L chain (1:3000), anti-chicken .gamma. chain (1:3000) and 
anti-chicken .mu. chain (1:3000) mouse monoclonal antibodies (ascites) 
were prepared and used as the primary antibody, peroxidase (HRPO)-labeled 
goat anti-mouse IgG (1:300) was used as the secondary antibody, and 
dimethylaminobenzene (DAB) was used as the substrate. 
II-5. Results 
As to two clones of MuH1 and MuH4, expression of cIg was examined. As a 
result, expression of .mu. chain was found in MuH1, and expression of .mu. 
chain and L chain was found in MuH4, as shown in Table 1. Since chicken 
immunoglobulins appeared in the above two clones, in order to examine 
their producibility, chicken immunoglobulins in the culture supernatant of 
each cell were detected by the ELISA method and the western blotting 
method. As a result, secretion of .mu. chain and L chain were found in the 
culture supernatant of MuH4, but secretion of chicken immunoglobulin was 
not found in the culture supernatant of MuH1, as shown in FIG. 1. The 
generation times of both clones were longer than those of R27H1 and R27H4 
by 2-3 hours (Table 1). 
Subsequently, sensitivities of MuH1 and MuH4 were examined to an 
aminopterin concentration of HAT medium. As shown by the results in FIG. 
2, in 0.5.times.10.sup.-7 M aminopterin concentration, MuH4 cells were 
completely dead 5 days from the start of culturing, and MuH1 cells were 
completely dead 2 days from the start of culturing, respectively. 
Aminopterin sensitivity of MuH1 cells was further examined to a low 
concentration. As a result, about 95% of cells were dead 4 days from the 
start of culturing, but a part of cells grew, in a 0.25 or 
0.1.times.10.sup.-7 M aminopterin concentration. 
TABLE 1 
______________________________________ 
Characteristics of Parental Cell Lines 
Ig Expression Generation 
Cytoplasmic Ig Time 
Cell Line 
L .mu. .gamma. 
Secretion 
(hr) 
______________________________________ 
R27H1 - + - .mu. 17.29 
R27H4 + + - IgM 13.80 
MuH1 - + - ND* 20.89 
MuH4 + + - IgM 15.72 
______________________________________ 
*Not detectable 
III. Cell Fusion of Human IgG-Immunized Chicken Spleen Cell with MuH1, MuH4 
as Patental Cell 
III-1. Immunized Chicken 
In the example, CB line white leghorn was used as the chicken for 
immunization. 
III-2. Antigen, Immunization Schedule 
Human IgG was used as the antigen, and 200 .mu.g/ml human IgG solution was 
prepared using PBS (-). Chickens of 4-6 weeks old were immunized 
intramuscularly with 100 .mu.g of the human IgG in 500 .mu.l PBS (-) 
emulsified with an equal volume of FCA. After 3-4 weeks from the primary 
immunization, the chickens were injected with 200 .mu.g of human IgG 
intravenously as a secondary immunization. Titer of chicken serum after 
each immunization was measured by the ELISA method (FIG. 3). 
III-3. Cell Fusion 
A. Preparation of Medium 
An IMDM medium containing 10% FBS was used as the medium for cell fusion. 
Hypoxantine, aminopterin and thymidine were added an IMDM medium 
containing 15% FBS at a concentration of 1.times.10.sup.-4 M, 
2.times.10.sup.-7 M and 1.6.times.10.sup.-5 M, respectively, and used an 
HAT medium. 
B. Preparation of Chicken Spleen Cell 
The chicken were sufficiently phlebotomized by drawing blood from the 
heart, and the spleen was taken out. The spleen was decapsulated, and 
roughly crushed by a scissors. The crushed matter was put in a glass 
homogenizer wherein about 10 ml of serum-free RPMI 1640 medium was added 
and the homogenizer was run 2-3 times to prepare a cell suspension. The 
suspension was passed through a stainless steel screen (#200 mesh), and 
centrifuged at 273 G for 5 minutes. The supernatant was completely 
removed, and about 40 ml of serum-free RPMI 1640 medium was added to the 
cell precipitates. In order to remove erythrocytes from the cell 
suspension, about 4 ml of the cell suspension was gently superposed on 
about 2 ml of Ficoll-Paque placed in a 15 ml centrifuge tube, and 
centrifuged at 715 G for 10 minutes. After centrifugation, cells located 
between the supernatant and the Ficoll-Paque were taken out, and suspended 
in a suitable amount of serum-free RPMI 1640 medium. The suspension was 
washed three times by centrifugation to prepare a chicken spleen cell 
suspension. 
C. Cell Fusion 
Parental cells were recovered in a 50 ml polypropylene centrifuge tube, and 
washed three times with a serum-free RPMI 1640 medium. The washed parental 
cells were mixed with the antigen-immunized spleen cells at a mixing ratio 
of 1:5, and centrifuged at 267 G for 5 minutes. The supernatant was 
removed completely by suction and agglutination of cells was loosened by 
tapping the bottom of the centrifuge tube lightly. Then, 1 ml of PEG 1500 
solution previously warmed to 38.degree. C. was gradually added while 
taking 1 minute. Meanwhile, the centrifuge tube was shaken laterally, and 
the suspension was stirred occasionally by the tip of a pipette. After 
adding the PEG solution, 10 ml of serum-free RPMI 1640 medium previously 
warmed to 38.degree. C. was added gradually while taking about 5 minutes. 
About 30 ml of the same medium was further added and centrifuged at 300 G 
for 5 minutes. The supernatant was gently removed by suction and the 
medium for hybridoma containing 10% FBS was added. 
The cell precipitates were loosened lightly, and 100 .mu.l (5 or 
8.times.10.sup.5 spleen cells/well) of the cell suspension was put into 
each well of a 96 well plate for tissue culture, and cultured at 
38.5.degree. C. in a 5% CO.sub.2 incubator. 
After 24 hours, 100 .mu.l of an HAT medium doubled in concentration was 
added to each well, and after the second day after cell fusion, the medium 
was changed every 2-4 days to an HAT medium having a normal concentration. 
From the 10-14th day, the medium was changed to an HT medium, and cultured 
for 1 week. Cells in some wells in the plate were cultured using a medium 
containing 2.times.10.sup.-5 M ouabain 7 days after the fusion. 
Hypoxanthine, thymidine and aminopterin concentration of the HAT medium 
were 1.times.10.sup.-4 M, 1.6.times.10.sup.-5 M and 
0.5-0.8.times.10.sup.-7 M, respectively. 
As shown by the results in Table 2, cell growth was found in all wells 
using R27H4, and the cells grown in most wells were small cells, possibly 
not hybridomas. 
In the case of MuH1, cells began to die the second day after HAT selection, 
and most cells were dead by the 4-5th day. On the other hand, in the case 
of MuH4, the cells first grew, but began to die by the 4th day, and most 
cells were dead by the 6-8th day. Hybridomas appeared in every well from 
about the 7-8th day, and the hybridomas grew stably. The fusion rate of 
MuH1 was 60.76% and 73.96% in the case of 5 or 8.times.10.sup.5 spleen 
cells/well, and that of MuH4 was 20.49% and 17.36%, respectively. Growth, 
of small cells which were possibly not hybridomas was not found at all. 
III-4. ELISA for Screening Hybridoma 
In order to detect specific antibodies, 50 .mu.l of human IgG, human IgG-Fc 
or human IgG-Fab diluted with PBS(-) at a concentration of 1 .mu.g/ml was 
put into each well of an ELISA plate for ELISA, and immobilized by 
allowing it to react at 4.degree. C. overnight. The ELISA plate was washed 
5 times with PBS (-) containing 0.05% Tween-20 (Tween-PBS), and 200 .mu.l 
of PBS (-) containing 1% gelatin was added to each well, followed by 
allowing it to react at 37.degree. C. for 1 hour. After washing with 
Tween-PBS, 50 .mu.l of the supernatant of the wells wherein hybridomas 
appeared was added to each wells of the plate. After allowing it to react 
at 37.degree. C. for 2 hours, the plate was washed with Tween-PBS. 50 
.mu.l of HRPO-goat anti-chicken IgG (1:3000) diluted 3000 times with Tris 
buffer solution (TBS) was added to each well, and allowed to react at 
37.degree. C. for 1 hour. After washing with Tween-PBS, 100 .mu.l of 
substrate solution containing phenylenediamine was added to each well, and 
allowed to react for 30 minutes at room temperature under dark conditions. 
50 .mu.l of 2M H.sub.2 SO.sub.4 was added to each well to terminate the 
reaction. Then, the absorbance at 490 nm was measured. 
III-5. Western Blotting for Detection of Specificity 
Specificity of the culture supernatant of hybridomas which indicated 
positive by the ELISA method was examined by the western blotting method. 
Each sample was mixed with human IgG-Fc and human IgG-Fab and 2-ME buffer 
solution, and boiled at 100.degree. C. for 5 minutes. 10% acrylamide gel 
was used for SDS-PAGE. 0.1 .mu.g of human IgG-Fc and 0.3 .mu.g of human 
IgG-Fab were applied to each lane of a minislab gel, and electrophoretic 
migration was conducted. The human IgG-Fc was previously purified by 
Protein G Sepharose 4 (Pharmacia) . 
After the electrophoretic migration, migrants were transfered to a 
nitrocellulose membrane and allowed to react with the culture supernatant 
of each hybridoma, HRPO-goat anti-chicken IgG (1:3000) was used as the 
secondary antibody and then the color was developed by DAB. In addition, 
HRPO-goat anti-human IgG (1:3000) was allowed to react as a control. 
III-6. Results of Screening Hybridoma 
Hybridomas which produced specific antibodies were screened by the ELISA 
method. As shown in the results by Table 2, exhibition of hybridomas 
having antibody-producing ability was found in hybridomas derived from 
MuH1 and MuH4. The production of antibodies of the hybridomas was very 
stable and it was successful in obtaining clones having antibody-producing 
ability from all hybridomas subjected to cloning by the soft agar culture. 
The specificity of the antibodies produced by the clones was examined by 
the ELISA method and the western blotting method. 
TABLE 2 
______________________________________ 
Comparison of Fusion Rate among Parental Cell Lines 
Number of Wells 
Number of Wells 
Exhibiting Exhibiting 
Parental 
Cell Growing Specific Antibody 
Cell Line 
(%) (%) 
______________________________________ 
Exp. 1 R27H4 288/288 (100) 
1/288 (0.01) 
MuH1 175/288 (60.76) 
7/175 (4.00) 
MuH4 59/288 (20.49) 
2/59 (3.39) 
Exp.2 R27H4 288/288 (100) 
2/288 (0.01) 
MuH1 213/288 (73.96) 
11/213 (5.16) 
MuH4 50/288 (17.36) 
7/50 (14.00) 
______________________________________ 
TABLE 3 
______________________________________ 
Specificities of Chicken Monoclonal Antibodies 
ELISA (O.D. 492 nm) 
Western Blotting 
mAb IgG IgG-Fc IgG-Fab IgG-Fc IgG-Fab 
______________________________________ 
CHM1-HuIg1 
0.407 0.043 0.502 - + 
CHM1-HuIg2 
0.407 0.467 0.031 + - 
CHM1-HuIg3 
0.419 0.504 0.029 + - 
CHM1-HuIg4 
0.467 0.438 0.092 + - 
CHM4-HuIgl 
0.389 0.458 0.021 + - 
CHM4-HuIg2 
0.352 0.398 0.081 + - 
CHM4-HuIg3 
0.465 0.459 0.035 + - 
CHM4-HuIg4 
0.412 0.478 0.0026 + - 
______________________________________ 
As shown in the results in Table 3, among the antibodies of the 8 
hybridomas, 7 antibodies exhibited reactivity with human IgG-Fc, and the 
other antibody exhibited reactivity with human IgG-Fab. In the western 
blotting, all 7 antibodies, which exhibited reactivity with human IgG-Fc 
by the ELISA method, reacted with a 50 k daltons protein which is human 
IgG-Fc, but the reactivity with human IgG-Fab was not found. The other 
antibody (CHMI-HuIg1) reacted with human IgG-Fab (about 50 k daltons), but 
not with 50 k daltons protein of human IgG-Fc, and reacted with a 
molecular weight of 100 k daltons protein or more. Two antibodies 
(CHM1-HuIg3 and CHM4-HuIg1) which exhibited the reactivity with human 
IgG-Fc and the antibody (CHM1-HuIg1) which exhibited the reactivity with 
human IgG-Fab were subjected to western blotting, and the results were 
shown in FIG. 4. 
IV. Properties of Hybridomas Fused by MuH1 or MuH4 Cells and Human 
IgG-Immunized Chicken Spleen Cells 
IV-1. ELISA for Detection of Specificity of Chicken Immuonogloblin Type 
Specificity of chicken immnoglobulin type was detected by the ELISA method. 
In order to detect the specificity of the antibodies, 50 .mu.l of human 
IgG diluted with PBS(-) at a concentration of 1 .mu.g/ml was put into each 
well of a ELISA plate, and immobilized by allowing it to react at 
4.degree. C. for 1 day. After allowing a culture supernatant of each 
hybridoma to react, the aforementioned anti-chicken L chain (1:5000), 
anti-chicken .gamma. chain (1:50000) and anti-chicken .mu. chain (1:50000) 
mouse monoclonal antibodies (ascites) were used as the secondary 
antibodies, and then, HRPO-goat anti-mouse IgG (1:3000) was used as the 
tertiary antibody. 
IV-2. Reactivity with Human IgG 
As to chicken anti-human IgG antibodies produced by 8 hybridomas derived 
from MuH1 or MuH4, their types of chicken immunoglobulins were examined by 
the ELISA method. As shown by the results in Table 4, all antibodies 
having .gamma. chains and L chains produced by the 8 clones exhibited 
reactivity with human IgG, but the antibody having .mu. chains did not 
exhibit reactivity with human IgG at all. 
TABLE 4 
______________________________________ 
Comparison of Ig Type of Antibodies 
Produced by Hybridomas 
ELISA (O.D. 492 m) 
Hybridoma Anti L Chain 
Anti .gamma. Chain 
Anti .mu. Chain 
______________________________________ 
CHM1-HuIg1 0.68 0.892 -0.011 
CHM1-HuIg2 0.712 0.861 -0.003 
CHM1-HuIg3 0.703 0.842 -0.009 
CHM1-HuIg4 0.544 0.769 -0.011 
CHM4-HuIgl 0.502 0.632 -0.014 
CHM4-HuIg2 0.354 0.525 0.017 
CHM4-HuIg3 0.672 0.694 -0.015 
CHM4-HuIg4 0.397 0.655 -0.013 
______________________________________ 
IV-3. ELISA for Quantification of Chicken Monoclonal Antibodies 
The concentration of immunoglobulin secreted from the hybridomas was 
determined by the ELISA method. Each culture supernatant to be tested was 
prepared by culturing antibody-producing hybridoma cells in an IMDM medium 
containing 10% FBS and 5.times.10.sup.-5 M or no ouabain in a 
concentration of 5.times.10.sup.5 cells/ml, and then culturing at 
38.5.degree. C. for 24 hours in a 5% CO.sub.2 incubator. 
On the ELISA plate, goat anti-chicken IgM-Fc (1:500, 50 .mu.l/well) was 
immobilized for the measurement of the concentration of chicken IgM or 
goat anti-chicken IgG-Fc (1:500, 50 .mu.l/well) was immobilized for the 
measurement of the concentration of chicken IgG. After blocking by Block 
Ace, the ELISA plate was allowed to react with 50 .mu.l of each hybridoma 
culture supernatant (dilution ratio, 1:10.about.1:1280) and chicken IgM 
(50 .mu.l/well) or chicken IgG (50 .mu.l/well) diluted with PBS (-) to 
1-300 ng/ml as a control. The aforementioned anti-chicken .mu. chain 
(1:100,000) and anti-chicken .gamma. chain (1:10,000) mouse monoclonal 
antibodies were used as the secondary antibodies, and HfRPO-goat 
anti-mouse IgG (1:3,000) was used as the tertiary antibody. 
IV-4. Quantification of Immunoglobulins in Chicken Hybridomas Culture 
The quantification of immunoglobulins in chicken hybridomas culture was 
examined by the ELISA method. Hybridomas stably growing in a medium 
containing ouabain were used for the examination of antibody-producing 
ability in the medium containing ouabain. As shown by the results in Table 
5, although the concentration of immunoglobulin was decreased in the 
medium containing ouabain compared with an ouabain-free medium, the 
decrease was not so remarkable in most hybridomas. The concentration of 
immunoglobulin of MuH1-derived hybridomas was greater than MuH4-derived 
hybridomas. The concentration of IgG from MuH4-derived hybridomas was less 
than 1 .mu.g/ml, but 3 hybridomas among 4 MuH1-derived hybridomas produced 
more than 2 .mu.g/ml IgG under ouabain-free conditions. Particularly, 
CHM1-HuIg 3, which did not express a .mu. chain, produced IgG in a great 
amount of more than 5 .mu.g/ml. On the other hand, the concentration of 
IgM from MuH1-derived hybridomas was similar to the concentration of IgG, 
but the concentration of IgM from MuH4-derived hybridomas was 1-4 times as 
much as the concentration of IgG. 
TABLE 5 
______________________________________ 
Concentration of IgG and IgM Produced by 
Parental Cells and Their Hybridomas 
IgG (.mu.g/ml) 
IgM (.mu.g/ml) 
No Oua No Oua 
Cell Line 
Added Oua Added Added Oua Added 
______________________________________ 
MuH1 0 0 0 0 
MuH4 0 NT* 0.26 NT 
CHM1-Hulg1 
2.38 1.58 3.12 2.07 
CHM1-Hulg2 
3.96 3.48 3.18 2.4 
CHM1-Hulg3 
5.22 5.92 0 0 
CHM1-Hulg4 
0.24 0.19 0.26 0.26 
CHM4-Hulg1 
0.72 0.57 1.53 1.2 
CHM4-Hulg2 
0.45 0.31 1.98 1.47 
CHM4-Hulg3 
0.65 0.26 0.78 0.76 
CHM4-Hulg4 
0.75 0.27 2.07 0.29 
______________________________________ 
*Not tested