Anti-human papillomavirus monoclonal antibody, hybridoma producing the same and process for preparing the same

An anti-human papillomavirus (HPV) monoclonal antibody reactive with various types of HPVs, which is produced by a hybridoma comprising a fused cell of a mouse spleen cell from a mouse immunized with an alkali-treated HPV type 1 (HPV-1) and a mouse myeloma cell, and which is reactive with polypeptides of about 57 kilodaltons, about 160 kilodaltons and about 230 kilodaltons of HPV-1, and a process for preparing the same. The monoclonal antibody of the invention is reactive with various types of HPV and useful for diagnosis of various types of HPV infection (primary diagnosis).

This invention relates to an anti-human papillomavirus (HPV) monoclonal 
antibody, a hybridoma producing said monoclonal antibody and a process for 
preparing said monoclonal antibody. More particularly, this invention 
relates to an anti-HPV monoclonal antibody reactive with various types of 
HPVs, which is produced by a hybridoma comprising a fused cell of a mouse 
spleen cell from a mouse immunized with an alkali-treated HPV type 1 
(HPV-1) and a mouse myeloma cell, and which is reactive with polypeptides 
of about 57 kilodaltons, about 160 kilodaltons and about 230 kilodaltons 
of HPV-1, a hybridoma producing said monoclonal antibody and a process for 
preparing said monoclonal antibody. The anti-HPV monoclonal antibody of 
this invention is useful for diagnosis of various types of HPV infection. 
TECHNICAL BACKGROUND AND PRIOR ART 
Human papillomavirus (HPV) is a small DNA virus infectious to human beings 
which belongs to papovaviridae and is classified into various types based 
on base sequences of DNA thereof. Hitherto, there have been isolated and 
identified more than 50 types of HPV from HPV-infected tissues. 
HPV infection causes a variety of diseases such as verruca vulgaris, 
verruca plana, myrmecia, epidermodysplasia verruciformis, condyloma 
acuminatum, laryngenal papillomatosis, etc. which are distinguished 
depending on regions of human the body at which the symptom appears, and 
features on shapes and tissue images thereof. Since some of these diseases 
become malignant and are turned into cervical cancer, squamous carcinoma 
and the like, a great attention has focused on the early diagnosis of HPV 
infectious disease. The HPV infectious disease mentioned herein is meant 
to include not only the various above mentioned diseases induced by the 
HPV infection but also conditions where HPV is carried by individuals 
without outbreak of disease. 
HPV infection can be diagnosed by detecting an antigen-antibody reaction 
between an antigen in epidermal tissues taken from individuals suspected 
of the infection and an anti-HPV antibody. 
In diagnosis of HPV infection utilizing the anti-HPV antibody, it is 
preferable to firstly diagnose whether there is any HPV infection by 
utilizing an antibody capable of reacting with various types of HPV 
(primary diagnosis) and then to determine the type of infecting HPV with a 
type-specific antibody which reacts only with a specific type of HPV 
(secondary diagnosis). A diagnosis utilizing only the type-specific 
antibody without primary diagnosis requires a large number of 
type-specific antibodies and wastes much time and labor since the 
diagnosis with the type-specific antibody requires all of more than 50 
type-specific antibodies and a large amount of epidermal tissues necessary 
for diagnosis using each type-specific antibody. 
Among known antibodies capable of reacting with various papillomaviruses 
(hereinafter referred to as "PV") is a rabbit anti-bovine papillomavirus 
(hereinafter referred to as "BPV") antibody which is a known reagent 
commercially available from DAKO CO. (catalogue No. B580). This antibody 
is obtained by immunizing a rabbit with a chemically treated bovine 
papillomavirus type 1 (hereinafter referred to as "BPV-1") and it has been 
evaluated to be capable of reacting with any of a wide variety of PVs not 
only BPV-1. However, the present inventors have tested this antibody for 
its reactivity with a variety of PVs and have found that this antibody 
could not detect some HPV infectious diseases as will be shown in the 
Experiment described hereinafter. 
Another known antibody reactive with a variety of HPV is an anti-HPV 
antibody produced by immunizing a rabbit with a sodium dodecyl sulfate 
(hereinafter referred to as "SDS")-disrupted HPV (JNCI Vol. 64, pp495-500, 
1980). However, it is necessary to steadily provide a large amount of HPV 
as an antigen for producing this antibody steadily but HPV grows only in 
human body and a method has not yet been established for growth of HPV in 
tissue culture and the like, and hence, this antibody is disadvantageous 
to an industrial, steady production in a large amount. 
It is known that a monoclonal antibody, which is produced by forming a 
hybridoma of an antibody-producing cell and a cell capable of rapidly and 
semi-permanently proliferating such as myeloma cell, followed by culture 
of said hybridoma, is more advantageous than a polyclonal antibody from 
various points of view. 
A production of an anti-HPV monoclonal antibody by a hybridoma is disclosed 
in European Patent Publication No. 174228A and Roseto A. et al. (J. Gen. 
Virol. Vol. 65, pp 1319-1324, 1984). The anti-HPV monoclonal antibodies 
disclosed in these references are, however, type-specific antibodies 
capable of reacting only with human papilloma-virus type 1 (HPV-1) and not 
those reactive with various other HPVs. According to the description of 
the literature, Western blotting of the monoclonal antibodies revealed 
that they reacted with 54 kilodaltons and 76 kilodaltons of polypeptides 
in the protein component of HPV-1. 
U.S. Pat. No. 4,551,270 and PCT Application WO 8605816A also disclose an 
amino acid sequence of a short peptide which can be a common antigen of 
various HPVs and describe that a monoclonal antibody capable of reacting 
with various HPVs can be prepared by utilizing the peptide as an antigen. 
However, an anti-HPV monoclonal antibody capable of reacting with various 
HPVs is not actually obtained in these references. 
Lex M. Cowsert et al. (JNCI, Vol. 79, No. 5, pp1053-1057, Nov. 1987) also 
report a monoclonal antibody produced by immunizing a mouse with an 
SDS-treated bovine PV; and Orth G. et al. (Virology No 1. 91, pp243-255, 
1978) disclose a polyclonal antibody produced by immunizing a guinea pig 
with an alkali-treated HPV, said polyclonal antibody being genus specific, 
not type-species specific. 
A novel monoclonal antibody capable of reacting with a variety of HPVs and 
a novel process for preparing said monoclonal antibody are still earnestly 
desired in the field. 
BRIEF SUMMARY OF THE INVENTION 
Under such circumstances, the present inventors have intensively studied to 
develop an anti-HPV monoclonal antibody capable of reacting with various 
HPVs which is useful for primary diagnosis of HPV infection, and as a 
result, have found that the monoclonal antibody can be obtained by 
alkali-treating HPV-1 to prepare an antigen, preparing a hybridoma 
producing an antibody against said antigen, cloning said hybridoma and 
selecting those hybridomas producing a monoclonal antibody reactive with 
various HPVs and that the monoclonal antibody thus prepared is useful for 
primary diagnosis of HPV infection. 
An object of the invention is to provide a novel anti-HPV monoclonal 
antibody which is reactive with all types of HPVs and is useful for 
primary diagnosis of HPV infection. Another object of the invention is to 
provide a method for preparing said anti-HPV monoclonal antibody by a cell 
fusion technology. These and other objects and advantages of the invention 
will be apparent to those skilled in the art from the following 
description.

DETAILED EXPLANATION OF THE INVENTION 
The novel anti-HPV monoclonal antibody reactive with various types of HPVs 
of this invention is produced by a hybridoma comprising a fused cell of a 
mouse spleen cell from a mouse immunized with an alkali-treated HPV type 1 
(HPV-1) and a mouse myeloma cell, and which is reactive with polypeptides 
of about 57 kilodaltons, about 160 kilodaltons and about 230 kilodaltons 
of HPV-1, a hybridoma producing said monoclonal antibody and a process for 
producing said monoclonal antibody. 
The method of the invention for producing the monoclonal antibody 
comprises: 
a) treating HPV-1 with an alkali to prepare an antigen, 
b) fusing a spleen cell from a mouse immunized with said antigen and a 
mouse myeloma cell to produce a hybridoma, 
c) cloning said hybridoma and selecting those hybridomas producing desired 
anti-HPV monoclonal antibody, and 
d) allowing said hybridomas to grow in a culture medium or within the 
peritoneal cavity of a mouse, harvesting the culture supernatant or the 
mouse ascites and separating the desired antibody therefrom. 
The hybridoma of the invention producing the monoclonal antibody has been 
internationally deposited on Feb. 9, 1989 at the Fermentation Research 
Institute Agency of Industrial Science and Technology, Tsu Kuba Science 
City, Ibaraki 305, Japan under Budapest Treaty under the accession number 
FERM BP-2278, which is designated "anti-human papillomavirus monoclonal 
antibody (K1H8) producing hybridoma". 
Preparation of Hybridoma 
The hybridoma of the invention is prepared by the following steps (A) to 
(E). 
(A) Preparation of Antigen 
Epidermal tissue infected with HPV-1 is suspended in a buffer, e.g. 
phosphate buffered saline (hereinafter referred to as "PBS"), ground with 
a grinder and centrifuged to remove cellular components to give a crude 
HPV-1. 
The above crude HPV-1 is added to an aqueous cesium chloride solution and 
the mixture is subjected to density gradient centrifugation and the 
portion of a band having a density of 1.34 g/cm.sup.3 is collected to give 
an HPV-1 solution. This HPV-1 solution is then dialyzed against PBS to 
give a solution of purified HPV-1. 
The purified HPV-1 is then treated with an alkali to prepare an 
alkali-treated HPV-1 (antigen). The alkali treatment can be made by 
dialyzing the solution of the purified HPV-1 against an alkaline aqueous 
solution of pH 10 to 11, preferably pH of about 10.5 at about 4.degree. C. 
for 10 to 20 hours. The alkaline aqueous solution used in the alkali 
treatment is preferably a buffer containing 2-aminoethanol. 
(B) Preparation of Antibody Producing Spleen Cell 
Mice (e.g. BALB/c mice, preferably of more than 6 weeks old) are immunized 
with an emulsion comprising the above alkali-treated HPV-1 (antigen) 
together with an adjuvant such as Freund's adjuvant and spleen is taken 
from the animals to prepare antibody producing spleen cells. 
The immunization is usually carried out by administering the alkali-treated 
HPV-1 (antigen) to the animal at least 3 times. A dose in each 
administration is 1 to 1000 .mu.g/animal of the alkali-treated HPV-1. The 
alkali-treated HPV-1 is preferably administered to mice in the form of an 
emulsion comprising an aqueous solution having a concentration of 100 to 
1000 .mu.g/ml of HPV-1 in admixture with an equivalent volume of an 
adjuvant (e.g. Freund's adjuvant). 
After immunization, spleen is taken out from mice and spleen cells are 
dispersed into Dulbecco's modified Eagle's minimum medium (hereinafter 
referred to as "DMEM medium") to prepare a suspension of antibody 
producing spleen cells. 
(C) Preparation of Myeloma Cells 
8-Azaguanine resistant myeloma cells, for example, commercially available 
mouse-derived myeloma cell P3X63Ag8U.1 (hereinafter referred to as 
"P3U1"), Sp2/0-Ag14, P3X63-Ag8.653 etc. (all these cells are commercially 
available from Dainippon Pharmaceutical Co., Ltd.) are employed. 
All these cells are cultured in a culture medium (e.g. RPMI 1640 medium 
supplemented with 5 to 20% fetal calf serum or DMEM medium supplemented 
with 5 to 20% fetal calf serum) containing about 100 .mu.M 8-azaguanine at 
37.degree. C. under atmosphere containing 5 to 10% CO.sub.2 and washed 
with a medium deficient in 8-azaguanine for use in the subsequent cell 
fusion. 
(D) Cell Fusion and Selection of Anti-HPV Antibody Producing Hybridomas 
The antibody producing spleen cells and the myeloma cells prepared above 
are cell-fused and those hybridomas producing an anti-HPV antibody are 
selected. 
The cell fusion is carried out by mixing a suspension of the antibody 
producing spleen cells and a suspension of the myeloma cells, subjecting 
the mixture to a low speed centrifugation to remove supernatant to give a 
mixture of both antibody producing cells and myeloma cells, adding thereto 
a polyethylene glycol (hereinafter referred to as "PEG") solution, 
stirring and shaking the mixture as described in Nature, Vol. 266, p550, 
1977, or by mixing the antibody producing cells, the myeloma cells and a 
PEG solution together and subjecting the mixture to a low speed 
centrifugation in accordance with the method described in Somat. Cell 
Genet., Vol. 3, p231, 1977. 
PEG preferably has an average molecular weight of 1000 to 6000 and is 
preferably used as a solution in DMEM medium at a concentration of 
preferably 30 to 50% (w/w). 
A mixing ratio of the antibody producing cells to the myeloma cells is 
preferably such that the amount of the antibody producing cells is 1 to 20 
times larger than that of the myeloma cells. 
For selection of hybridomas, the mixture of cells obtained by the above 
cell fusion is poured into each well of microplate, cultured in a medium 
where only hybridomas can grow, e.g. HAT medium (comprising hypoxanthine, 
aminopterin and thymidine), and selecting wells where cells grow to give 
hybridomas. 
The culture is carried out by suspending the cell mixture obtained by the 
cell fusion in HAT medium at about 1.times.10.sup.6 cells/ml, pouring the 
suspension into each well of microplate and culturing it at 37.degree. C. 
under atmosphere containing 5 to 10% CO.sub.2 for 10 to 14 days while 
replacing the medium with a fresh medium usually on the fourth day. 
The anti-HPV antibody producing hybridomas are then selected by enzyme 
immunoassay of the culture supernatant of each hybridoma. 
(E) Cloning of Hybridomas and Selection of Hybridomas of the Invention 
The anti-HPV antibody producing hybridomas prepared in (D) are cloned by a 
limiting dilution method with HAT medium or other conventional method and 
those hybridomas being capable of producing an anti-HPV monoclonal 
antibody are selected by the enzyme immunoassay as in (D). 
The anti-HPV monoclonal antibody producing hybridomas thus cloned are 
subjected to selection by the tissue staining procedure as described in 
the following Experiment where plural epidermal tissues infected with 
various HPVs are employed, and the anti-HPV monoclonal antibody producing 
hybridoma reactive with all of these tissues is selected. 
The thus selected hybridoma is cultured in HT medium (medium containing 
hypoxanthine and thymidine) at 37.degree. C. under atmosphere containing 5 
to 10% CO.sub.2 for about 1 to 2 weeks and then subcultured in a usual 
medium, for example RPMI 1640 medium supplemented with 5 to 20% fetal calf 
serum or DMEM medium supplemented with 5 to 20% fetal calf serum under the 
same conditions, and then stored. 
The hybridoma is stored by suspending them in either RPMI 1640 medium or 
DMEM medium, both media being supplemented with 5 to 20% fetal calf serum 
and further being added with about 10% dimethyl sulfoxide (hereinafter 
referred to as "DMSO") under cooling with liquid nitrogen. 
The thus obtained hybridoma producing the antibody reactive with all HPVs 
of the invention (designated "anti-human papillomavirus monoclonal 
antibody (K1H8) producing hybridoma") has been deposited under the 
accession number FERM BP-2278 as mentioned hereinbefore. 
Preparation of Anti-HPV Monoclonal Antibody 
The anti-HPV monoclonal antibody of the invention can be prepared by 
culturing the hybridoma prepared as mentioned above at 37.degree. C. under 
atmosphere containing 5 to 10% CO.sub.2 followed by separation and 
purification from the culture supernatant. 
The hybridoma is cultured in RPMI 1640 medium supplemented with 5 to 20% 
fetal calf serum or DMEM medium supplemented with fetal calf serum for 3 
days to 3 weeks, preferably for 10 to 14 days while subculturing about 
every about 3 days. 
Alternatively, the anti-HPV monoclonal antibody of the invention can be 
prepared by growing the hybridoma prepared as mentioned above in the 
peritoneal cavity of small mammals compatible with the hybridoma such as 
mice (e.g. BALB/c mice), followed by separating and purifying from the 
ascites. The intraperitoneal growing of the hybridoma is usually carried 
out for 1 to 3 weeks. 
The anti-HPV monoclonal antibody of the invention can be purified by 
centrifugation of the culture solution or the ascites, followed by salting 
out with ammonium sulfate or ion exchange chromatography. 
Salting out is preferably carried out with a 30 to 50% saturated aqueous 
solution of ammonium sulfate. The monoclonal antibody salted out is then 
dialyzed against PBS to give a PBS solution containing the purified 
monoclonal antibody of the invention. 
Ion exchange chromatography is preferably conducted by column 
chromatography with an anion exchange resin such as DEAE Sepharose 
(manufactured by Pharmacia, Sweden). The elution is preferably carried out 
with Tris buffer (pH about 7.0). 
The aqueous solution of anti-HPV monoclonal antibody of the invention thus 
obtained can be stored after freezing or lyophilization. 
The anti-HPV monoclonal antibody of the invention can be used for diagnosis 
of HPV infection. 
Diagnosis of HPV infection can be made by detecting the reactivity of the 
anti-HPV monoclonal antibody of the invention and epidermal tissues or 
cells taken from individuals (tissue staining). 
A tissue staining procedure can be conducted either by a direct procedure 
wherein the anti-HPV monoclonal antibody of the invention labelled with a 
labelling material such as biotin or fluorescein isothiocyanate 
(hereinafter referred to as "FITC") etc. is reacted with tissues or cells 
from individuals and the label is detected as a measure of the reactivity, 
or by an indirect procedure wherein the anti-HPV monoclonal antibody of 
the invention is reacted with tissues or cells from individuals and the 
reacted monoclonal antibody is then reacted with a secondary antibody 
labelled with a labelling material such as biotin or FITC, followed by 
detection of the label as a measure of the reactivity. 
When the label is biotin, the tissues or cells are stained by an 
avidin-biotinylated peroxidase complex method ("Handbook of Experimental 
Immunology", Vol. 4, ed. by D. M. Weir, BLACKWELL SCIENTIFIC PUBLICATIONS, 
129.10) and the reactivity is detected by microscopic observation. Brown 
stained particles are observed in those tissues or cells infected with 
HPV. 
In case of labelling with FITC, the reactivity is detected by observation 
with a fluorescence microscope. Green fluorescent particles are observed 
in those tissues or cells infected with HPV. 
The anti-HPV monoclonal antibody of the invention can detect various HPVs. 
For example, the anti-HPV monoclonal antibody of the invention clearly 
reacts with tissues infected with various HPVs such as myrmecia, verruca 
plana, condyloma acuminatum and the like, and thereby, the HPV infection 
of all samples can be tested (see Experiment described hereinbelow), which 
the conventional rabbit anti-BPV antibody could not detect the HPV 
infection in some samples. Thus, the anti-HPV monoclonal antibody of the 
invention is quite useful for diagnosis of HPV, particularly for diagnosis 
of primary diagnosis of HPV. 
Experiment (Diagnosis of HPV Infection) 
A diagnostic test was conducted using the anti-HPV monoclonal antibody of 
the invention which is subjected to an antigen-antibody reaction with 
tissues. The antigen-antibody reaction was detected by the 
avidin-biotinylated peroxidase complex method as mentioned above with 
staining of tissues. At the same time, commercially available rabbit 
anti-BPV antibody was also tested for comparison. 
1. Materials 
(a) Specimen 
The following 9 tissue specimens from individuals infected with HPV were 
used. 
______________________________________ 
Myrmecia one specimen 
Verruca plana one specimen 
Condyloma acuminatum 
seven specimens (a) to (g) 
______________________________________ 
(b) Antibody 
Antibody of the Invention 
An aqueous solution of the anti-HPV monoclonal antibody at a concentration 
of 2 .mu.g/ml which was prepared by diluting the monoclonal antibody 
solution obtained in Example 2 with PBS. 
Reference Antibody 
An aqueous solution of a rabbit anti-BPV antibody at a concentration of 10 
.mu.g/ml which was prepared by diluting the rabbit anti-BPV antibody 
(manufactured by DAKO CO., catalogue No. B580) with PBS. 
2. Method 
Specimens taken from individuals were embedded in paraffin and sliced into 
5 .mu.m pieces of width. The paraffin embedded tissue sections were each 
put on a slide glass and paraffin was removed in the conventional manner 
and thereto was added the antibody solution (0.2 ml), and the mixture was 
reacted at room temperature for 2 hours and washed with PBS (pH 7.4) at 
4.degree. C. three times each for 5 minutes. 
Then, to the specimens were added dropwise biotin-labelled anti-mouse 
IgG+IgA+IgM (H+L)(0.2 ml) (manufactured by ZYMED Laboratories, Inc.) 
adjusted to a concentration of 5 .mu.g/ml with PBS (pH 7.4) in case of the 
anti-HPV monoclonal antibody of the invention, or biotin-labelled 
anti-rabbit IgG (0.2 ml)(manufactured by ZYMED Laboratories, Inc.) 
adjusted to a concentration of 5 .mu.g/ml with PBS (pH 7.4) in case of the 
rabbit anti-BPV antibody, and the mixture was reacted at room temperature 
for 1 hour and washed with PBS (pH 7.4) at 4.degree. C. three times each 
for 5 minutes. 
The avidin-biotinylated peroxidase complex solution (0.2 
ml)(Vectastain.RTM., manufactured by Vector Laboratories, Inc.) was then 
added dropwise to the specimens, and the mixture was reacted at room 
temperature for 30 minutes and washed with PBS (pH 7.4) at 4.degree. C. 
three times each for 5 minutes. 
Then, to the specimens were added dropwise each 0.2 ml of PBS (pH 7.4) 
containing 3,3'-diaminobenzidine.4HCl (0.2 mg/ml) and 0.015% (w/v) 
hydrogen peroxide, and the mixture was reacted at room temperature for 30 
minutes and the reaction was quenched with addition of distilled water. 
Finally, one to two drops of a PBS solution containing 50% (v/v) glycerol 
was added to the specimens and, after covering with a cover glass, 
developed color images were observed with a microscope. Those specimens 
taken from individuals infected with HPV revealed brown stained particles. 
The staining was graded as follows: 
______________________________________ 
+: 1 to 5 stained particles 
++: 6 to 30 stained particles 
+++: Not less than 31 stained particles 
-: No stained particles 
.+-.: Unclearly stained particles 
______________________________________ 
3. Results 
The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Antibody (Ab) 
Ab of the 
Rabbit 
invention 
anti-BPV 
Specimens (K1H8) Ab (Control) 
______________________________________ 
Myrmecia ++ +++ 
Verruca plana +++ ++ 
Condyloma acuminatum (a) 
++ +++ 
Condyloma acuminatum (b) 
+ - 
Condyloma acuminatum (c) 
+ .+-. 
Condyloma acuminatum (d) 
++ ++ 
Condyloma acuminatum (e) 
+ + 
Condyloma acuminatum (f) 
+++ +++ 
Condyloma acuminatum (g) 
+++ +++ 
______________________________________ 
As is clearly shown in Table 1, the anti-HPV monoclonal antibody of the 
invention could detect all of the HPV infectious diseases tested while the 
commercially available rabbit anti-BPV antibody could not detect some HPV 
infectious diseases. 
This invention is more specifically illustrated by the following Examples 
but should not be construed to be limited thereto. In Examples, percentage 
means % (v/v) unless otherwise mentioned. 
EXAMPLE 1 
(a) Preparation of Hybridoma) 
The hybridoma of the invention was obtained by the following procedures (a) 
to (e). 
(a) Preparation of Antigens 
Wart tissue infected with HPV-1 (5 g) was added to PBS (pH 7.4; 50 ml), 
ground with a grinder and centrifuged at a low speed (1000 rpm; 10 
minutes). The resultant supernatant was then centrifuged at a high speed 
(10,000 rpm; 10 minutes) to give precipitate of crude HPV-1. The obtained 
crude HPV-1 was then overlayed on an aqueous solution of cesium chloride 
(density: 1.34 g/cm.sup.3) and subjected to a density gradient 
centrifugation (30,000 rpm; 20 hours) with SW41Ti Rotor (manufactured by 
Beckmann CO.,). A band having a density of 1.34 g/cm.sup.3 was separated 
and dialyzed against PBS (1000 ml) at 4.degree. C. for 16 hours to prepare 
a solution of a purified HPV-1 in PBS (1 ml). 
A mixture of 5 M NaCl (12 ml), 2M Tris(hydroxymethyl)aminomethane-HCl (pH 
7.5; 4 ml) and 250 mM ethylenediamine tetraacetate (hereinafter referred 
to as "EDTA"; 1.6 ml) was diluted with distilled water to make a total 
amount of 500 ml and pH thereof was adjusted to 10.5 by addition of 
2-aminoethanol to prepare a dialysis solution. The purified HPV-1 prepared 
as mentioned above was dialyzed against this dialysis solution at 
4.degree. C. for 16 hours, followed by dialysis against a dialysis 
solution comprising 1 mM EDTA-10 mM Tris(hydroxymethyl)aminomethane -HCl 
(pH 8.0; 500 ml) at 4.degree. C. for 16 hours to give 1 ml of a solution 
(density; 500 .mu.g/ml) of an alkali-treated HPV-1 (antigen). 
(b) Preparation of Antibody Producing Spleen Cells 
The alkali-treated HPV-1 solution (250 .mu.l; containing about 125 .mu.g of 
the alkali-treated HPV-1) was mixed with an equivalent volume of complete 
Freund's adjuvant (manufactured by Difco Laboratories, Inc.) to prepare an 
emulsion. Female BALB/c mouse of 9 weeks old was subjected to the primary 
immunization by administering the emulsion subcutaneously (490 .mu.l) and 
at footpads (10 .mu.l). After 14 days, the animal was boostered with an 
emulsion comprising the alkali-treated HPV-1 and incomplete Freund's 
adjuvant in the same manner. Fourteen days after the booster injection, 
the animal was given intravenous administration of a physiological saline 
solution (200 .mu.l) of the above alkali-treated HPV-1 (about 10 .mu.g) 
for final immunization. After 3 days, the mouse was sacrificed and the 
spleen was aseptically taken out. The spleen was cut with scissors in DMEM 
medium and passed through a mesh to prepare a suspension of a single cell. 
The resultant suspension was washed with DMEM medium (.times.3) to prepare 
a suspension of the antibody-producing spleen cells in DMEM medium (10 ml; 
containing 9.2.times.10.sup.7 cells). 
(c) Preparation of Myeloma Cells 
Mouse myeloma cells P3U1 (5.times.10.sup.6 cells) were added to 25 ml of 
the following RPMI 1640 medium supplemented with fetal calf serum 
(hereinafter referred to as "RPMI medium") and thereto was added 
8-azaguanine (100 .mu.M). The cells were cultured under atmosphere 
containing 5% CO.sub.2 at 37.degree. C. for 5 days and washed with DMEM 
medium (.times.2) to prepare a suspension of the mouse myeloma cells P3U1 
in DMEM (10 ml; containing 1.4.times.10.sup.8 cells). 
RPMI 1640 medium supplemented with fetal calf serum was prepared by adding 
distilled water to a mixture of RPMI 1640 (manufactured by GIBCO; 10.4 g), 
sodium hydrogen carbonate (1.3 g), L-glutamine (25.2 mg), penicillin G 
(63.5 mg), streptomycin sulfate (100 mg), tylosin (10 mg), 
2-mercaptoethanol solution (manufactured by WAKO PURE CHEMICAL INDUSTRIES; 
40 .mu.l) and fetal calf serum (manufactured by FLOW Laboratories; 100 ml) 
to make a total amount of 1000 ml and passing through a 0.45 .mu.m 
membrane filter (manufactured by TOYO ROSHI CO., LTD) for sterile 
filtration. 
(d) Cell Fusion and Selection of Anti-HPV Antibody Producing Hybridomas 
The suspension of the antibody producing spleen cells (10 ml; 
9.2.times.10.sup.7 cells) prepared in the above procedure (b) and the 
suspension of the myeloma cells P3U1 (0.66 ml; 9.2.times.10.sup.6 cells) 
prepared in the above procedure (c) were mixed in a 50 ml centrifugation 
tube and the mixture was centrifuged at 1000 rpm for 10 minutes to 
precipitate the mixture of both cells. After removal of the supernatant, 
to the mixture of both cells was added dropwise DMEM medium (0.5 ml) 
containing PEG 1000 (42.5 w/w %) and DMSO (15%) while mildly stirring over 
a period of 1 minute. 1 ml, 1 ml, 5 ml, 5 ml and 10 ml of DMEM medium was 
then added dropwise in this order while mildly stirring at room 
temperature each over a period of 1 minute to fuse the cells. The treated 
mixture was then centrifuged at 1000 rpm for 10 minutes and the 
supernatant was removed to give a mixture of the fused cells. 
For selection of hybridomas, the above mixture of the treated cells was 
suspended in HAT medium (RPMI medium comprising 100 .mu.M hypoxanthine, 
0.4 .mu.M aminopterin and 16 .mu.M thymidine) to prepare a cell suspension 
(104 ml, cell number: about 10.sup.6 /ml). 
The above cell suspension was then placed in a 96-well microplate (Falcon 
3072 manufactured by FALCON) in an amount of 0.2 ml/well and the cells 
were cultured at 37.degree. C. under atmosphere containing 5% CO.sub.2. 
The cells were cultured for totally 10 days while replacing the whole 
supernatant in each well with fresh HAT medium (0.2 ml/well) on the fourth 
day so that the hybridomas can grow sufficiently. 
The culture supernatant (hereinafter referred to as "culture supernatant 
(A)") in each well was then subjected to enzyme immunoassay as hereinbelow 
described to select anti-HPV antibody producing hybridomas. 
Enzyme Immunoassay for Selection of Anti-HPV Antibody Producing Hybridomas: 
The alkali-treated HPV-1 solution (104 .mu.l) prepared in the above 
procedure (a) was diluted to a concentration of 1 .mu.g/ml with 0.05M 
sodium carbonate-sodium hydrogen carbonate buffer (pH 9.6) and 100 .mu.l 
of the diluted solution was poured into each well of 96-well microplate 
(Immulon 600 manufactured by Greiner Labortechnik) and allowed to stand at 
4.degree. C. overnight to fix the alkali-treated HPV-1 to the well. After 
washing each well with PBS (pH 7.4) containing 0.05% Tween-20 
(polyoxyethylene sorbitan monolaurate) (hereinafter referred to as 
"T-PBS"), to the well was added PBS (pH 7.4; 300 .mu.l) containing 0.5% 
(w/v) bovine serum albumin (manufactured by WAKO PURE CHEMICAL INDUSTRIES; 
hereinafter referred to as "BSA") and the mixture was allowed to stand at 
room temperature for 1 hour, followed by removal of the supernatant. Each 
of the above culture supernatant (A) was diluted twofold with PBS (pH 7.4) 
containing 0.1% (w/v) BSA and 100 .mu. l thereof was added to each well at 
room temperature, and the mixture was reacted for 2 hours and washed with 
T-PBS (pH 7.4). 
Biotin-labelled anti-mouse IgG+IgA+IgM (H+L) (manufactured by ZYMED 
Laboratories, Inc.) was diluted to a concentration of 5 .mu.g/ml with PBS 
(pH 7.4) containing 0.1% (w/v) BSA, and 100 .mu.l thereof was added to 
each well, and the mixture was reacted at room temperature for 1 hour and 
washed with T-PBS (pH 7.4). 
Streptavidin peroxidase (manufactured by Amersham International PLC) was 
then diluted 1000-fold with PBS (pH 7.4) containing 0.1% (w/v) BSA, and 
100 .mu.l thereof was added to each well, and the mixture was reacted at 
room temperature for 30 minutes and washed with T-PBS (pH 7.4). 
To each well was then added 100 .mu.l of 0.15M citrate-sodium phosphate 
buffer (pH 5.0) containing 0.015% (w/v) hydrogen peroxide and 
o-phenylenediamine (0.2 mg/ml) as a substrate to react at room temperature 
for 5 minutes. 50 .mu.l of 5N sulfuric acid was then added to each well to 
quench the reaction to give a test solution (hereinafter referred to as 
"test solution (T)"). 
On the other hand, the same procedures were repeated except that HAT medium 
was employed in place of the culture medium (A) to give a reference 
solution (hereinafter referred to as "test solution (C)"). 
Absorbance at 492 nm was measured for the above test solutions (A) and (C) 
using Corona 2 wave length microplate photometer (MTP-22, manufactured by 
CORONA ELECTRIC CO., LTD). Fifteen wells which contained the test solution 
(T) showing 0.1 or more higher absorbance than that of the test solution 
(C) were selected to obtain 15 kinds of anti-HPV antibody producing 
hybridomas. 
(e) Cloning of Hybridomas and Selection of Hybridoma of the Invention 
The above anti-HPV antibody producing hybridomas were cloned by a limiting 
dilution method. 
The anti-HPV antibody producing hybridomas selected in the procedure (d) 
and BALB/c mouse thymus cells (obtained from BALB/c mouse of 6 weeks old 
by a conventional procedure) were suspended in HAT medium to prepare a 
suspension of mixed cells (concentration of hybridomas: 3 cells/ml, 
concentration of the thymus cells: about 3.times.10.sup.6 cells/ml). 0.2 
ml of the suspension was poured into each well of 96-well microplate 
(Falcon 3072) and the cells were cultured at 37.degree. C. under 
atmosphere containing 5% CO.sub.2 for 14 days. Those wells which produced 
one colony per well were selected and the culture supernatant thereof was 
subjected to enzyme immunoassay as in the above procedure (d) and three 
clones of hybridomas were selected which produced a monoclonal antibody 
having a high reactivity. 
Three antibodies (designated K1H8, K3B5 and K4B2, respectively) produced by 
the above selected hybridomas were tested for their reactivity with the 
alkali-treated HPV-1 as described in the procedure (a) by the method 
described in the procedure (d). The results are shown in Table 2 with the 
absorbance (A.sub.492) 
TABLE 2 
______________________________________ 
Antibody Reactivity (absorbance A.sub.492) 
______________________________________ 
K1H8 0.89 
K3B5 0.81 
K4B2 0.72 
Control 0.01 
______________________________________ 
In order to select hybridomas which produce monoclonal antibodies reactive 
with various HPVs for these three clones, they were tested for the 
antigen-antibody reaction with a plurality of HPV-infected tissues in 
accordance with the test method described in Experiment. Among three 
clones, the antibody (K1H8) from one clone reacted all of these tissues 
tested, and hence, this hybridoma was selected. 
The hybridoma producing this antibody K1H8 was grown in the manner 
described hereinbelow and deposited under accession number FERM BP-2278 as 
mentioned hereinbefore. 
EXAMPLE 2 
(Preparation of Monoclonal Antibody) 
The anti-human papillomavirus monoclonal antibody (K1H8) was prepared from 
the hybridoma culture supernatant. 
The above "anti-human papillomavirus monoclonal antibody (K1H8) producing 
hybridomas" (10.sup.5 cells) in HAT medium were transferred into HT medium 
(RPMI medium containing 100 .mu.M hypoxanthine and 16 .mu.M thymidine; 5 
ml) and subcultured at 37.degree. C. under atmosphere containing 5% 
CO.sub.2 for 14 days, and then transferred into RPMI medium (100 ml) and 
cultured at 37.degree. C. under atmosphere containing 5% CO.sub.2 for 14 
days. 
The hybridomas were separated and removed by centrifugation (1000 rpm, 10 
minutes) to give the culture supernatant (100 ml). To the supernatant was 
added a saturated aqueous solution of ammonium sulfate (66.7 ml), the 
mixture was stirred at room temperature for 1 hour, allowed to stand at 
the same temperature for 1 hour, centrifuged (10,000 rpm) at 4.degree. C. 
for 20 minutes and the supernatant was discarded to give precipitate. 
To the precipitate was added 0.9% (w/v) physiological saline solution (100 
ml) to dissolve and the solution was centrifuged (10,000 rpm, 20 minutes) 
at 4.degree. C. to give supernatant. To the supernatant was added a 
saturated aqueous solution of ammonium sulfate (50.0 ml), and the mixture 
was stirred at room temperature for 1 hour, allowed to stand at the same 
temperature for 1 hour, centrifuged (10,000 rpm, 20 minutes) at 4.degree. 
C. and the supernatant was discarded to give precipitate. 
The obtained precipitate was dissolved in PBS (100 ml) and the solution was 
dialyzed against PBS (5 liters) at 4.degree. C. for 16 hours to give a 
solution of the anti-human papillomavirus monoclonal antibody (K1H8) in 
PBS (100 ml, concentration: 23.5 .mu.g/ml). 
The antigen analysis test (A) described hereinbelow proved that this 
monoclonal antibody had characteristics capable of inducing 
antigen-antibody reaction with polypeptides of about 57 kilodaltons, about 
160 kilodaltons and about 230 kilodaltons of HPV-1. 
Further, the immunological classification test (B) described hereinbelow 
proved that this antibody belonged to immunoglobulin subclass G.sub.1 and 
the L chain thereof had isotype of .kappa. chain. 
(A) Antigen Analysis Test of Anti-human Papillomavirus Monoclonal Antibody 
(K1H8) 
The HPV-1 purified in Example 1-(a) was diluted to a concentration of 10 
.mu.g/ml and subjected to SDS-polyacrylamide gel electrophoresis in 
accordance with the procedure by Laemmli (see Nature, Vol. 227, pp680, 
1970). After electrophoresis, polyvinylidene fluoride membrane 
(manufactured by Millipore) was put on the gel and electrophoresis was 
further conducted in a transfer buffer (comprising 20% methyl alcohol, 25 
mM Tris(hydroxymethyl)aminomethane-HCl, and 192 mM glycine, pH 8.3) at 
4.degree. C. and at 15 V for 16 hours for transcription. After 
transcription, the polyvinylidene fluoride membrane was added to PBS (pH 
7.4; 10 ml) containing 5% (w/v) BSA, and the mixture was subjected to 
blocking at 37.degree. C. for 1 hour. 
A solution of the monoclonal antibody (K1H8) was prepared at a 
concentration of 1 .mu.g/ml with PBS (pH 7.4) containing 1% (w/v) BSA, and 
to 10 ml of this solution was added the above blocked polyvinylidene 
fluoride membrane and the mixture was incubated at 37.degree. C. for 2 
hours, followed by washing (.times.5) with T-PBS (pH 7.4) each for 5 
minutes to give monoclonal antibody (K1H8)-reacted polyvinylidene fluoride 
membrane. 
Then, to a solution (10 ml) of biotin-labelled anti-mouse IgG+IgA+IgM 
(H+L)(manufactured by ZYMED Laboratories, Inc.) diluted to a concentration 
of 5 .mu.g/ml with PBS (pH 7.4) containing 1% (w/v) BSA was added the 
above polyvinylidene fluoride membrane, and the mixture was incubated at 
37.degree. C. for 1 hour, followed by washing (.times.5) with the same 
T-PBS (pH 7.4) as above to give biotin-labelled anti-mouse IgG+IgA+IgM 
(H+L)-reacted polyvinylidene fluoride membrane. 
To a solution (10 ml) of streptavidin peroxidase (manufactured by Amersham 
International PLC) diluted to 1000-fold with PBS containing 1% (w/v) BSA 
was then added the above polyvinylidene fluoride membrane, and the mixture 
was incubated at 37.degree. C. for 30 minutes, followed by washing 
(.times.5) with T-PBS (pH 7.4) each for 5 minutes to give streptavidin 
peroxidase-reacted polyvinylidene fluoride membrane. 
The above polyvinylidene fluoride membrane was then added to PBS (pH 7.4; 
10 ml) containing 3,3'-diaminobenzidine.4HCl (0.2 mg/ml) and 0.015% (w/v) 
hydrogen peroxide, and the mixture was reacted at room temperature for 10 
minutes. After the reaction was quenched with distilled water, the 
polyvinylidene fluoride membrane was dried with a drier to reveal bands of 
polypeptides of HPV-1 reacted with the monoclonal antibody (K1H8). 
Each molecular weight of the polypeptides of HPV-1 reacted with the 
monoclonal antibody (K1H8) was determined with commercially available 
molecular weight markers (catalogue No. 17-0445-01 manufactured by 
Pharmacia and catalogue No. 161-0305 manufactured by Bio-Rad) in the 
following manner. 
That is, the commercially available molecular weight markers were subjected 
to the same SDS-polyacrylamide gel electrophoresis as above and 
transcribed onto polyvinylidene fluoride membrane, followed by staining 
with an aqueous solution containing 0.25% (w/v) Coomassie Brilliant Blue R 
250, 45% ethanol and 45% acetic acid at room temperature for 10 minutes. 
The membrane was then decolorized with 90% aqueous ethanol solution at 
room temperature for 5 minutes and bands of the molecular weight marker 
polypeptides were observed. A migrating distance of each band was then 
measured to give a relative mobility. A molecular weight of each HPV-1 
polypeptide reacted with the above monoclonal antibody (K1H8) was 
determined from a relative mobility of each band of the HPV-1 
polypeptides. 
(B) Immunological Classification Test 
In order to determine the class, subclass and isotype of the L chain of the 
above monoclonal antibody (K1H8), the following procedures were performed. 
That is, rabbit antibodies against each class and subclass of mouse 
immunoglobulin (IgA, IgM, IgG.sub.1, IgG.sub.2a, IgG.sub.2b and IgG.sub.3) 
and rabbit anti-mouse L chains (.kappa. chain and .lambda. chain) 
antibodies (manufactured by Miles Laboratories, Inc.) were used to prepare 
solutions in 0.05M sodium carbonate-sodium hydrogen carbonate buffer (pH 
9.6), each solution containing 5 .mu.g/ml of one of the antibodies. 100 
.mu.l of the solutions was poured into each well of 96-well microplate 
(Immulon 600) and the wells were allowed to stand at 4.degree. C. 
overnight for fixation. After washing each well with T-PBS (pH 7.4), PBS 
(pH 7.4; 300 .mu.l) containing 0.5% (w/v) BSA was added to each well and 
allowed to stand at room temperature for 1 hour, followed by removal of 
the supernatant. 
Then, the monoclonal antibody (K1H8) solution (0.5 ml) obtained above was 
diluted twofold with PBS (pH 7.4) containing 0.1% (w/v) BSA and 100 .mu.l 
of the solution was added to each well, and the mixture was reacted at 
room temperature for 2 hours, followed by washing with T-PBS (pH 7.4). 
100 .mu.l of a solution of biotin-labelled anti-mouse IgG+IgA+IgM 
(H+L)(manufactured by ZYMED Laboratories, Inc.) diluted to a concentration 
of 5 .mu.g/ml with PBS (pH 7.4) containing 0.1% (w/v) BSA was then added 
to each well, and the mixture was reacted at room temperature for 1 hour, 
followed by washing with T-PBS (pH 7.4). 
100 .mu.l of streptavidin peroxidase (manufactured by Amersham 
International PLC) diluted 1000-fold with PBS (pH 7.4) containing 0.1% 
(w/v) BSA was then added to each well, and the mixture was reacted at room 
temperature for 30 minutes, followed by washing with T-PBS (pH 7.4). 
Then, to each well was added 100 .mu.l of 0.15M citrate-sodium phosphate 
buffer (pH 5.0) containing 0.015% (w/v) hydrogen peroxide and 
o-phenylenediamine (0.2 mg/ml) as a substrate, and the mixture was reacted 
at room temperature for 5 minutes. 
To each well was then added 50 .mu.l of 5N sulfuric acid to quench the 
reaction. Absorbance at 492 nm was measured for the above reaction 
solutions with Corona 2 wave length microplate photometer (MTP-22 
manufactured by CORONA ELECTRIC CO., LTD) to assess the reactivity of the 
monoclonal antibody (K1H8) of the invention with each immunoglobulin. The 
highest reactivity of the anti-human papillomavirus monoclonal antibody 
(K1H8) with rabbit anti-mouse IgG.sub.1 antibody and rabbit anti-mouse 
.kappa. antibody proved that the anti-human papillomavirus monoclonal 
antibody (K1H8) belonged to immunoglobulin subclass G.sub.1 wherein 
isotype of L chain is a .kappa. chain. 
EXAMPLE 3 
The hybridoma was grown in the peritoneal cavity of a mouse and the 
anti-human papillomavirus monoclonal antibody (K1H8) was obtained from 
ascites. 
Pristane (2,6,10,14-tetramethylpentadecane; 0.5 ml) was intraperitoneally 
administered to a BALB/c mouse 9 weeks old. Twenty one days after the 
administration, anti-human papillomavirus monoclonal antibody (K1H8) 
producing hybridomas, which were prepared by culturing the anti-human 
papillomavirus monoclonal antibody (K1H8) producing hybridomas prepared in 
Example 1 in HT medium in the manner described in Example 2 followed by 
subculture in RPMI medium, were suspended in RPMI medium (concentration: 
about 2.times.10.sup.7 cells/ml) and 0.5 ml of the suspension was 
intraperitoneally administered to the animal. 
Ascites produced (about 10 ml) was then taken out and subjected to 
centrifugation (1000 rpm, 10 minutes) to precipitate cellular components 
to give supernatant (7.4 ml). To the supernatant was added a saturated 
aqueous solution of ammonium sulfate (4.9 ml) and the mixture was stirred 
at room temperature for 1 hour, allowed to stand at the same temperature 
for 1 hour, subjected to centrifugation (10,000 rpm, 20 minutes) at 
4.degree. C. and the supernatant was discarded to give precipitate. 
To the precipitate was added 0.9% (w/v) physiological saline solution (7.4 
ml) to dissolve and the solution was subjected to centrifugation (10,000 
rpm, 20 minutes) at 4.degree. C. to give supernatant. To the supernatant 
was added a saturated aqueous solution of ammonium sulfate (3.7 ml) and 
the mixture was stirred at room temperature for 1 hour, allowed to stand 
at the same temperature for 1 hour, subjected to centrifugation (10,000 
rpm, 20 minutes) at 4.degree. C. and the supernatant was discarded to give 
precipitate. 
The precipitate was dissolved in PBS (7.4 ml) and the solution was dialyzed 
against PBS (3000 ml) at 4.degree. C. for 16 hours to give a solution of 
the anti-human papillomavirus monoclonal antibody (K1H8) in PBS (7.4 ml, 
concentration: 7.01 mg/ml). The thus obtained monoclonal antibody showed 
the same reactivity against the polypeptides of HPV-1 as that of the 
monoclonal antibody prepared in Example 2.