Note: Descriptions are shown in the official language in which they were submitted.
3~'V7~7~;;
ADULT T-C~LL Lli'UKEL'lIA ANTIGENS,
METHOD FOR Tl:IEIR PREPARAT:[ON ~ND
E'OR ASSAYING ANTIBODIES TEIERETO
BAC~GROUND OF THE :[NVENTION
This lnvention relates to an adult T-cell
leukemia antigen, a method for its prepara-tion and a
method for assaying an antibody associated with adult T-
cell leukemia and, ~ore particularly, to a me-thod ~or
assaying an anti~ody according to indirect fluorescent
antibod~ technique or enzyme-antibody technique usin~ an
antiyen which ~pecificall~ reacts with sera of patien-ts
with adult T cell leukemia.
Adult T-cell leukemia ~hereinafter referred to
as ATL) is a malignant disease which attacks adults, bu-t
the cause of the disease has not yet been clari~ied
comple-tely. ~See, Takatsuki, K., Uchiyama, T., Sagawa,
K. and Yodoi, ~, (1977) in "Topics in ~ematology", eds.
Seno, S~, Takaku, F., and ~rino, S. (Excepta Medica,
Amsterdam), pp. 73-77 and Uchiyama, T., Yodoi, J.,
Sagawa, K., Takatsuki, K. and Uchino, H., Blood 50,
481 49~ (1977), etc.)
Based on the knowledge tha-t cultured ATI. cells
or ATL virus-associated cell lines contain an antigen
specific to ATL (~TI,-associated antigen or ATL-antigen,
herei~after referred to as ATLA) which is not cletected
in ~resh ATL cells in peripheral bloods of ATL pa-tients,
and tha~ sera of ATL patients or of certain healthy
2~ adults in ATL-endemic areas contain an an-tibody whic
specifically reac-ts ~Jith sai.cl ~TL~ (hereinafter referred
to as ATLA antibody), there has recently been proposed a
method for assayiny ~TLA antibody us:ing fluorescel~ce
i~munoassay whieh is capable of cliaynosin~ ATL. (Proc~
Natl. cad. Sci. U.S.A., Vol 78, No. 10, pp. 6476-6480
(1981)).
. This method comprises plaeiny ATL-posi.tive
eells as an antigen on a glass slide, reactin~ the
antiyen with a test sample serum and fluorescenee labeled
anti-human i~munoglobulin ~, and mieroseopieally obc;erv-
iny loealization of the fluoreseence marker to assay
ATLA antibody~
However, this method has various disadvanta~es
sinee it uses living eells per se as an.antiyen and it is
necessary to conduct mieroseopieal observation for eaeh
sample (test sample serum), resulting in that not only
operation is eomplicated but also it is difficult to
assay and diaynose a large amoun-t of tes-t samples with
ease and rapidly~
Further, the above method is disadvantageous
in that it is difficult to avoid the fluetuation in
antigen positivity since intact cells are used and,
therefore, it is difficult to ob-tain a desired quality
of antigen eonstantly, whieh would leacl -to erroneous
assay and diagnosis. Therefore, this method does not
meet the requirement that assay ean be earried ou-t uncler
~1~ 7 ~ o ~
fixed conditions, which requirement needs to be ScltiS-
fied when wide prevalence of the techni~ue concerned is
attemp-ted.
SU~M~R~ OE' TEIE INVENTION
It is an object of the presen-t inventiorl to
provide a method for asSayinCJ ATI,A antibody which
obviates the defects of the conventional method for
assaying ATLA an-tibody.
As a result of extensive investigation it has
now been ound that soluble cytoplasmic protein of
cultured ATL cells ~hereina~-ter referred to as SCP) and
solubilized protein of ATL virus (hereinafter referred
to as VAPl can be used as an antigen which can specifi-
cally react with ATLA antibody and that a larye amount
of test samples can be assayed with ease and rapidly as
well as under fixed conditions with high efficiency when
an insolubilized antiyen is used which is prepared by
insolubilizing SCP or VAP on an insoluble support.
The present invention is based on the abo-ve
findings and provides a method for asSayinCJ ATLA antibody
according to fluorescence immunoassay o~ enzyme immuno-
assay comprising usiny an insolubilized antigen compris-
ing an insoluble support haviny insolubilizecl thereon at
leas-t one substance selected from the group consistlng
25 of ~ soluble cytoplasmic protein of ATI, cells and a
solubilized protein o~ ATL virus.
-- 3 -
~'777~
Accordiny to -the present invention, a larye
amount of test samples can be assayed rapidly wlth
simplified operation; for example, sarnples rnore than
abou-t 10 -times as much as those ~1hi.ch can be treated
with conventional microscopical observation can be
assayed or the same amount of samples can be assayed
more than about 10 times as rapidly as the conventional
method .
BRIEF D~SCRIPTION OF THE DRAWIN5
Figure 1 is a cJraph plo-ttiny against dilution
of sera optical density at 492 nm of ATLA antibody
determined according to the method of the present inven-
tion (Example 5).
DETAILED ~ESCRXPTION OF THE INVENTION
As the culture of ATL cells to be utilized as
the source of SCP in the present lnvention, there are
illustrated ~TL cells already established as cell line
(Miyoshi, I., Kubonishi, I., Sumida, M., E~iraki, S.,
Tsubota, T., Kimura, I., Miyamoto, K. and Sato, J.,
20 _ann 71, 155-156 (1980)~, culture of ATL cells separated
fr~n the peripheral blood or lymph node of patients haviny Al'L in a
conventional manner, and Ari$ viru~s-associated ce]ls
obtained by co-culturing the above-described ATL cells
and human T-cells (e.y., Nature 294, p. 770-771 (1981)~.
The human T--cells -to be utilized in the above-described
11~'77'~G
co-culturing are not particularly limi-ted, and there are
illustrated, for example, various T-cel:Ls from peripheral
blood, bone marrow, lymph node, spleen, tonsil, thyrnus,
etc.
Culturing of the above-described ATL cells or
co-culturing of the ATL cells and the human T-cells can
be conducted in an ordinary manner. ~edium is not
particularly limited, and any of various nutrient media
used for ordinary cell culture can be used. Preferred
examples o~ media include, for example, RP~I 16~0 medium
(~low Laboratories, Co.~ and minimum Eagle essential
medium (MEM medium) modified with a supplementary serum
such as fe-tal bovine serum (FCS) or calf serum.
Culturiny conditions are not particularly
different from those employed in ordinary cell culture,
and a temperature of about 36 ko about 38C and a pH of
about 6.4 to 7.6 are generally employed. If desired,
growth accelerators such as T-cell growth factor (TCGF)
or chemicals known as inducers of retrovirus of-ma~mals,
such as 5-iodo-2'-deoxyuridine (IdUrd~, cyclohexylimide
(CH), puromycin, phorbol 12-myristate 13-acetate (TPA),
n-butyrate (sodium n-butyrate), etc.l can be added.
Culturiny is advan-tageously conducted by exchanginy the
solution every 3 to 5 days, thereby allowiny desired
cells to yrow favorably.
'7'~
Further, as the SC~ a preparation can he used
which is obtained by hornoyenizing the above cultured ATL
cells in an approprlate bufEer solution such as physio~
logical saline, phosphate buffered saline, etc., ancl
recoverlng the subs-tance as supernatan~ by means of
centrifuga-tion or a like suitable separatinCJ means~
Examples of the ATL virus which can be used as
the source of VAP include those isolated from the
culture fluicl of the above~described ATL cells in a
conventional manner. The isolation of the ATL virus can
be conducted using a conventional centrifugation
technique. It is pre-erred to use ~TL virus fur-ther
purified by density yradient method.
VAP can be ob-tained b~ solubilizing the ArrL
virus. Solubilization of ATL vixus can be carried out
with ease using ordinary solubiliziny agents.
Examples of solubiliziny agents include
various surEactants such as nonionic surfactants, e.g.,
~ "Triton X-100" (a trade ~m~for a product of T~ako Pure
Chemical Co., Ltd.), "NP~~0" (a trade~ffl~ for a product
of Shell Co.), diyitonin, urea, etc., and anionic
surEactants, e.y., sodium dodeGyl sulfate 1SDS~, etc.
There is no part;cular limi.ta-tion on the
method o~ solubil;.zation. ~lowevér, it is preferred to
conduct solubilization usiny a solubill-~incJ agent in an
amount of O.Olgo to that corresponcliny -to a micel1e forrn~
in~ point at a -temperature of about 0 to about 100C
for several minu-tes to about 6 hours. PreEerably, the
VAP thus ob-tained is iurther purified by ordinary
techniques such as centrifugation, dialysis, etc., and
then subjected to an anion exchange column chromatography
using DEAE-cellulose, DEAE~Sephadex, etc., to adsorb and
remove viral nucleic acid which may remain in VAP.
The insolubilized antigen which is used in the
present invention can be prepared by insolubilizing th~
SCP and/or VAP on an insoluble suppor-t. There is no
particular limitation on -the insoluble support to be
used and ordinary ones used in physical adsorp-tion
methods, more particularly, porous carriers of poly-
lS styrene, glass, polycarbonate, polypropylene, etc., canbe used.
Fixation of SCP and/or VAP on the insoluble
support can be carried out in a conventional manner.
For e~ample, an insoluble support and SCP and/or VAP are
addecl to a solution such as physiological saline,
phosphate buffer, etc., and the mixture is reacted at
about 0 to about 37C Eor abou-t 2 to about 24 hours.
It is preferred this reaction be effected under reduced
pressure. After completion of the reac-tion, adsorption
sites which remain in the insoluble support are sa-turated
Tr~le l~ ~rk
7'7~;
in a conventional manller, for examp:Le, witn 0.2~ gela-tln,
0.2~ bo~lne serum albu~lin (BSA), etc.
The insoluble antigen thus obtained i5 washed
with water. The washed preparation is stored in a dried
sta-te or in the buffer solution described above.
The method oE the present inven~ion can be
carried out ~y assaying -the label activity of a labeled
antigen-antibody complex according to ordinary fluores-
cence immunoassay or enzyme immunoassay using the
insoluble antigen. More particularly, it can be carried
out by adding a test sample serum optionally diluted to
the insoluble antigen to efect immune reaction there-
between, washing the resulting antigen-antibody complex,
reacting the complex with a labeling effector to label
the complex, and assaying the label activity of the
labeled complex in a conventional manner.
~ hus, in the present inven-tion, use oE the
insoluble antigen enables one to assay at a time a large
amount of test sample sera rapidl~ with ease and high
precision at fixed conditions.
As the test sample sera ~hat can be used, sera
separated from blood collected in a conventional manner
from an adult on whom assay of ATI,A antibody is desired
or diluted preparations of these sera dilu-ted with an
appropriate buffer solu~ion. The buffer solution to be
- 8 ~
~977'76
used is not limited particular:ly. Usual.ly, it :is prefer
red to use a phosphate buffer solution (p~ 7.4).
The immune reaction between the test sample
serum and the insoluble an-tigerl can proceed simply by
mixing them. Usually, the reaction can proceed at about
4 to about 37C for abou~ 0.5 to about 16 hours.
;Desirably, after completion of the reac-ti.on, the reaction
product is washed sufficiently with an appropria-te
buffer solution, preferably physiological saline or the
i10 buffer solution which is used for the clilution of the
test sample serum.
Labeling of the resulting antigen-antibody
complex with a labeling effec-tor which follows the
immune reaction can be carried ou~ by mixiny the antiyen~
1.5 antibody complex obtained with a labeling ef:Eector
cliluted with the same buffer solution as described above
and allowing the mixture to react at abou-t ~ to abou-t
37C for about 0.5 to about 16 hours. A:Eter completion
of the reaction, it is preferred to wash the resultin~
coMplex sufficient1y in a similar manner ~o the above~
As the labeling effector there can be used any
~, .
composite material composed of a substance capable oE
`specificall~ binding to an antihody and various en~yme
or fluorescein. Representative examples of enzymes
_ g _
;,~
:
7~
inclu~le various ordinary enzyme reagents such as
peroxidase (PO~), chymotrypsinogen, procarboxypeptidase,
glyceroaldehyde 3-phosphoric ac:id dehydrogenase, amylase,
phosphorylase, D-Nase, P-Nase, etc.
The fluoresceirl is exempllfied by orclinary
fluorescent dyes such as fluorescein isothiocyanate
(FITC), tetramethylrhodamine isothiocyanate (TRITC),
substituted rhodamine isothiocyanate ~XRITC), ~odamine B
isothiocyanate, dichlorotriazine fluorescein (DTAF), etc.
~xamples of the substance capable o~ specifi-
cally binding to an antibody include "Protein A'i ~Pro A,
a product of Pharmacia Co.) and anti--human immuno-
ylobulin G (hereinafter referred to as IgG~ antibody
such as sheep anti-human IgG antibody, rabbit anti-human
IgG antibody, goat anti-human IgG antibody, mouse anti-
human IgG antibody, rat anti-human IgG antibody, etc.
Various complèxes of the substance capable o
specifically binding to an antibody and the labeliny
substance are available commercially, and they can be
used as a labeling effector in the present invention.
On the other hand,' such complexes ma~ be
prepared freshly before use according to conven-tional
method as described by B.F. ERLANGER et al.: Ac-ta.~ndo
25 crinol. Suppl. 168, 206 (1972) and ~.1l. KAROL et al.:
Proc. N _ Acad. Sci. U.S.~. 57, 713 (1967)._ _ _
- 10
7~
More specifi.cally, when an enZyMe i~ used, this
enzyme and Pro A or anti~human IgG antibody are coupled
in a buffer solution (pM 4 to 6) in the presence of an
appropriate oxldizing agent such as NaIO~ at rooM tempe-
rature for 2 to 5 hours and then reduced with an appro-
priate reduciny agent such as NaB~
With respect to the amounts of the reagents
used, it is preferred that about 1 to about 3 moles of
enzyme and about 100 to 300 moles of oxidizing agent be
used per mole of Pro A ox anti-huraan IgG antibody.
Preferably, the reducing agentis used in an a~ount of
about 1 to about 2 moles per mole of the oxidizing agent.
When labelin~ effectors are pxepared UsinCJ a
fluorescein! the fluorescein is added to water or physi-
ological saline whose pH is 6 to 8 and the mixture isreacted with Pro A or anti-human IgG antibody at about
0C to room temperature for about 0.5 to about 3 hours
(cf. "~ei.ko Kotai lIo" (Fluorescent ~ntibody Method)
IkacJaku Jikkenho Koza, No. 4, 263-270 (1972), lst..Ed.,
Nakayarna Shoten Co., I.td., Tokyo). It is preferred th~t
the amount of fluorescein to be used be about 1/50 part
by weight per part by weight of Pro A or anti-human IgG
antibody.
-- 11 ~
~ 77 7~
In the presen-t invention, the insoluble antigen-
test sample serum (ATLA an-tibody)~labelincJ efEector
eomplex can be assayecl in a conventional manner appropri-
ately depending on the labeling substance in the labeling
effector used by determining the label activity (i.e.,
aetivity of enzyme or fluorescence). In this way, a
large amoun-t of test sample serum, ATL~ antibody, ean be
assayed with ease and rapldly.
The present invention will now be deseribed in
more detail by the following reference examples ancl
examples which, however, are not to be construed as
limiting the present inven-tion in any way.
EX~MPLE
Preparation of Insoluble Antigen
Blood ~20 mQ) collected from a patient having
ATI. (50--year~old, male, living in Nagasaki city) using
heparin was centrifuged in "Ficoll Pack" (produced by
Pharmacia Japan Co.) to obtain 5 x 107 cells of periph-
eral blood lymphocyte.
These cells were cultured at 37C for 3 days
in RPMI 1640/10% calf serum (~low Laborator;es Co.) at a
eell concentra-tion of 3 x 105 cells/m~. A portion of the
thus-obtained cells (6 x 10~ cellsl was homogenized in a
O.OS M phosphate buffer solution ~con-tainin~ 0.14 M NaC~,
pH 7.4, hereinafter referred to as PBS~ and centrifuged
- ~2 -
7~
for 1 hour (105,000 x ~). I'he supernatan-t was collected
and its protein con-tent was adjusted with PBS to
120 ~g/mQ (the protein conten-t was de-termined by
colorimetric method usiny "Tonein R -TP", a trademark
for reagent for quanti-tative determination of total
protein produced by Otsuka Assay La~oratories, Co.-, Ltd.)
to obtain an SCP solution.
To the SCP solution (140 m~) were added 700
pieces of polystyrene beads (diameter: 6.4 mm, produced
by Precision Plastic Co., Ltd., U.S.A.) previously
washed serially with an aqueous 0.1 N E~CQ SOlUti.OII, an
a~ueous 0.1 N NaOH solution and ethanol, and the mixture
was allowed to stand for 6 hours at room temperature
under suction using an aspirator, followed by filtration
to obtain insolubilized antigen. This was stored in a
0.05 M phosphate buffer solution containlns 0.2% gelatin
(pH 7.4) at 4C.
EX~MPLE 2
Prepara-tion of Insoluble Antigen
(1) ATL cells (Kyo-Ya cell obtained from Virus Research
Institute of Kyoto Univexsity) was cultivated a-t 37C
for ~ days in RP~I 1640/10 % calf serum ~ 50 ~g/mQ IdUrd
(Elow Laboratories Co.) at a cell concentration of
3 x 105 ceLls/mQ. The culture fluid was centrifuyed at
1,500 rpm for 10 minutes to separate cells and -the
culture medium, which were collected separately.
- 13 - ~
7~
(2) To a portion oE culturecl ~TL cells (5 ~ ]0 cells)
obtained in (1) above was added 30 mQ of physiological
saline and the mix-ture was homoyen:ized and then cen-tri-
fuyed at 105,000 x g for 1 hour. The supernatan-t was
collected and its protein content was adjustecl in the
same manner as in Example 1 to prepare an SCP solution
having a protein content o 120 ~g/mQ, from which an
insoluble antigen, i.e., antigen-adsorbirlc3 polystyrene
bead, was prepared in -the same manner as in E~ample 1.
The antigen obtained was allowed to stand for one night
in a 0.05 M phosphate buffer solution containing 0.2%
gelatin (pH 7.4), washed with water, dried and stored
till use~
(3) The culture medium (S00 rnQ~ obtained in (1~ above -
was centrifuged at 40,000 x g for 1 hour and the pellet
formed ~as collected. The pellet was subjected -to
density gradient cen-triEugation on 25 to 60~ sugar, and
fractions corresponding to density of 1.15 to 1.16 were
collected. These frac-tions were solubilized with 1;5 m~
of an aclueous 0.8 M MaCQ solution containiny 0.5~ Triton
X-100 at 4C for 60 minutes and then su~je~ced~-to centrifugation
at 35,000 rpm for 1 hour to collect supernatant.
The supernatant thus-obtained was dialyzed
against 0.02 M l'ris-~ICQ buffer solution containing 0.3
NaCQ (p~I 7~5) using a cellophane membrane for 5 hours to
14 - ~
remove solubilizing agen-t and adjust the concentra-tion
of salts. The VA~ solution thus obtained was passed
throuyh DEAE-cellulose colu~n equilibrated with the same
Txis-~lCQ buffer solution as above to obtain the fraction
which passed throuc;h the column withou~ stopping.
Distilled water was aclded to this fraction to adjust
its protein content to 2.8 ~g/mQ. To a ~0 m~ portion of
this solution were added 80 pieces o-f polystyrelle beads
described in Example 1 previously washed as in Example 1
and allowed to stand for 6 hours at room temperature to
obtain insoluble antigen. This was allowed to stand Eor
one night in a 0~05 P~I phosphate bu~fer solution con-tain-
ing 0.2~ gelatin (pH 7.4), washed with water, dried and
stored till use.
EXA~PLE 3
Preparation of Sample Serum
`; Blood samples were collected from a patient
.~
haviny ATL and a healthy adult usiny heparin, and
allowed to stand for 3 hours at room tempera-ture to
collect the supernatan-ts. Each of the supernatants was
centrifuged for 10 minutes at 2,0Q0 rpm to collect the
supernatants which were used as tes-t sa~plc sera.
EXAMPLE
; Assay of ~TLA Antibody
A series of diluted sera (80 x, 160 x, 320 x,
`, 640 x, 1,280 x) were prepared by double diluting each
~ - 15 -
.
..
,~
119 ~7~
test sample serurn prepared in Example 3 with a 0.05 M
phosvhate bufLer con.ainin~ 0.2% gelati.n (pH 7.4). To
0.5 mQ of each of the dilu'ced sample se.ra ~/as adcled one
piece of the insolu'Dle anti~en (an-igen-adsorbing
polys-ty.rene bead) prepared in Example 1 and allowed to
stand at 37C for 2 hours. The reactlon mixture was
removed by suction wi-h an aspirator, the bead was
washed with 2 mQ of physiological saline and the washin~
was removed by suction with an aspirator. -This procedure
was repeated three times.
Further, one piece of the bead thus-treated
was added to 0.55 mQ of peroxidase--labeled Protein A (a
product of E.Y. Laboratories Co.~ diluted wi-th the same
phosphate buffer as above to 4~,000 x and allowed to
stand at 37C for 2 hours followed by washing sufficient-
ly in the same manner as above.
On the other hand, E~2O2 was added with stirring
to 20 mQ of 0.2 M McLevin buffer solu-tion containing
60 my of o-phenylenediamine (pH 5.8) to adjust it to a
final concentration of 0.02 V/V % to prepare a coloring
agent.
In a tes-t tube, 2 mQ of physiological saline
and 0~5 mQ of the coloriny agent were brought and one
piece of bead prepared as described above was added
thereto. AEter allo~.~ing to stancl at room temperature
- 16 -
:
-\
~777~;
for 30 mi.nutes, 1 m~. of 3 N hyclrochloric acid was added
to stop the en~yma,ic reaction, and optical density at
~92 nm of t'ne reaction mlxture was measured.
The results obt,tined are shown in Table 1
below.
TABLE
Dilution of Test Sample Sera
x 80 x 160 x 320 x 6~0 x1280
ATL Patient
A 0~789 0~748 0.646 0.550 0.410
B 0.701 0.62d 0.550 0.423 0.356
. 10 C 0.558 0.449 0.367 0.291 0.222
Healthy Person
D 0.166 0.114 0.081 0.047 0.028
E 0.114 0.089 0.054 0.031 0.027
F 0.1~2 0.089 0.063 0.037 0.023
The same procedures as above were repea-tecl
except that instead of the insoluble antigen prepared in
Examp].e 1, the insoluble antigen (antigen-adsorbing
polystyrene beads) prepared in Example 2-(2) and Example
2-(3), respectively, were used for 80-fold or 100-old
diluted sera.
The results obtai.ned are shown in Table 2 and
; l'able 3 below.
- 17 -
7'ô'~
TABI,E 2
Insoluble Anti.gen Prepared in Example 2-(2)
Opti.ca] Density at 492 nm
:L00-F'old Diluted Seru~n
ATI, Patien-t
G 1.15~
~l 1.012
I 0.879
Healthy Person
J 0.108
K 0.09~1
L 0.011
.10 TABLF. 3
Insolub-le Antigen Prepared in Example 2-(3)
Optical Density a-t 492 ~n
80-Fold Diluted Serum
ATL Patient
M 0.826
N 0.722
1~ O 0.611
Healthy Person
P 0.23
0.329
18 -
'7'76
EX~PLE 5
For assaying ATLA antlbody, the same procedures
as ;n Exam~le 4 were repeated except -tha-t the antigen-
adsorbing polystyrene beads prepared in Example 2-(2)
as an insoluble antigen and 600-fold diluted peroxi.dase
labeled goat anti-human IgG (a product of E.Y. Labora-
tories Co.~ as a labeling effector were used. Optical
density at 492 nm was obtained for eac'n dilution of sera.
The resul.ts obtained are shown in Fi~ure 1 in
lQ ~hich curve 1 represents ATLA antibody level fo~ ATL
pati.ent~ and curve 2 that for healthy person.
As will be clear from the results shown in
Tables 1, 2 and 3 and Figure 1, accordiny to the method
of the present invention ATLA antibody can ke assayed
with ease by measuring optical density, ancl, therefore,
the present invention is useful for diagnosin~ and
screening ATL patients.
While the invention has been described in
detail and with reference to specific embodiments
thereof, it will be apparent to one skilled in the art
that various changes and modifications can be made
therein without depar-ting from the spirit and scope
thereof.
