CA1197776A - Adult t-cell leukemia antigens, method for their preparation and for assaying antibodies thereto - Google Patents
Adult t-cell leukemia antigens, method for their preparation and for assaying antibodies theretoInfo
- Publication number
- CA1197776A CA1197776A CA000426688A CA426688A CA1197776A CA 1197776 A CA1197776 A CA 1197776A CA 000426688 A CA000426688 A CA 000426688A CA 426688 A CA426688 A CA 426688A CA 1197776 A CA1197776 A CA 1197776A
- Authority
- CA
- Canada
- Prior art keywords
- adult
- antigen
- cell leukemia
- cell
- insolubilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
Abstract
ABSTRACT
An insolubilized antigen is disclosed which comprises an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen selected from the group consisting of a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus. Also, a process for pre-paring the insolubilized antigen and a method for assaying an antibody to adult T-cell leukemia associated antigen according to fluorescence immunoassay or enzyme immunoassay using the insolubilized antigen are disclosed. The use of the insolubilized antigen permits of speedy assay of the antibody in large amounts of test sample sera.
An insolubilized antigen is disclosed which comprises an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen selected from the group consisting of a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus. Also, a process for pre-paring the insolubilized antigen and a method for assaying an antibody to adult T-cell leukemia associated antigen according to fluorescence immunoassay or enzyme immunoassay using the insolubilized antigen are disclosed. The use of the insolubilized antigen permits of speedy assay of the antibody in large amounts of test sample sera.
Description
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
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.
~'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)._ _ _ 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
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.
~'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)._ _ _ 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 :
-\
~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.
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 '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.
- 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 :
-\
~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.
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 '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.
Claims (28)
1. An insolubilized antigen comprising an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen selected from the group consisting of a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus.
2. The insolubilized antigen as claimed in Claim 1, wherein said adult T-cell leukemia associated antigen is said soluble cytoplasmic protein immobilized on said insoluble support selected from the group consist-ing of polystyrene, glass, polycarbonate and polypropylene.
3. The insolubilized antigen as claimed in Claim 2, wherein said insoluble support is polystyrene beads.
4. The insolubilized antigen as claimed in Claims 2 or 3, wherein said soluble cytoplasmic protein is centrifugation supernatant of homogenate of said adult T-cell leukemia cell.
5. The insolubilized antigen as claimed in Claim 2, wherein said adult T-cell leukemia cell is that isolated from peripheral blood or lymph node collected from patient having adult T-cell leukemia.
6. The insolubilized antigen as claimed in Claim 5, wherein said adult T-cell leukemia cell is an established adult T-cell leukemia cell line.
7. The insolubilized antigen as claimed in Claim 2, wherein said adult T-cell leukemia cell is that transformed by co-cultivating an adult T-cell leukemia cell isolated from peripheral blood or lymph node collect-ed from a patient having adult T-cell leukemia with human T-cell.
8. The insolubilized antigen as claimed in Claim 7, wherein said human T-cell is derived from peri-pheral blood, bone marrow, lymph node, spleen, tonsil or thymus.
9. The insolubilized antigen as described in Claim 7, wherein said co-cultivation is carried out in an RPMI 1640 medium or a minimun Eagle essential medium modified with a fetal bovine serum or calf serum.
10. The insolubilized antigen as claimed in Claim 7, wherein said adult T-cell leukemia cell is an established adult T-cell leukemia cell line.
11. The insolubilized antigen as claimed in Claim 1, wherein said adult T-cell leukemia associated antigen is said solubilized protein immobilized on said insoluble support selected from the group consisting of polystyrene, glass, polycarbonate and polypropylene.
12. The insolubilized antigen as claimed in Claim 11, wherein said insoluble support is polystyrene beads.
13. The insolubilized antigen as claimed in Claim 11, wherein said adult T-cell leukemia virus is that purified by density gradient centrifugation.
14. The insolubilized antigen as claimed in Claim 13, wherein said solubilized protein is a protein obtained by solubilizing adult T-cell leukemia virus with a solubilizing agent.
15. The insolubilized antigen as claimed in Claim 14, wherein said solubilizing agent is a nonionic surfactant such as Triton X-100*, NP-40*, digitonin and urea or an anionic surfactant such as sodium dodesyl sulfate.
16. The insolubilized antigen as claimed in Claim 15, wherein said solubilizing agent is Triton X-100*.
17. A process for preparing an insolubilized antigen comprising an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen select-ed from the group consisting of a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus, which comprises mixing an adult T-cell leukemia associated antigen with an insoluble support in an aqueous medium, reacting them, saturating the adsorption sites which remain in said insoluble support and washing the resulting antigen with water.
18. The process as claimed in Claim 17, wherein
18. The process as claimed in Claim 17, wherein
Claim 18 continued said adult T-cell leukemia associated antigen is said soluble cytoplasmic protein immobilized on said insolble support selected from the group consisting of polystyrene, glass, polycarbonate and polypropylene.
19. The process as claimed in Claim 17, wherein said adult T-cell leukemia associated antigen is said solubi-lized protein immobilized on said insoluble support selected from the group consisting of polystyrene, glass, polycarbonate, and polypropylene.
20. A method for assaying an antibody to adult T-cell leukemia associated antigen according to fluoroescence immunoassay or enzyme immunoassay comprising reacting an insolubilized antigen as claimed in Claim 1 with a test sample serum and determining the resulting antigen-antibody complex.
21. The method as claimed in Claim 20, which further comprises reacting said antigen-antibody complex with a labeling effector capable of specifically binding an antibody.
22. The method as claimed in Claim 21, wherein said labeling effector is an enzyme- or fluoroescent-labeled substance capable of specifically binding an antibody selected from the group consisting of Protein A, sheep anti-human immunoglobulin G antibody, rabbit anti-human immunoglobulin G antibody, goat anti-human immunoglobulin G antibody, mouse anti-human immunoglobulin G
22. The method as claimed in Claim 21, wherein said labeling effector is an enzyme- or fluoroescent-labeled substance capable of specifically binding an antibody selected from the group consisting of Protein A, sheep anti-human immunoglobulin G antibody, rabbit anti-human immunoglobulin G antibody, goat anti-human immunoglobulin G antibody, mouse anti-human immunoglobulin G
Claim 22 continued...
antibody, and rat anti-human immunoglobulin G antibody.
antibody, and rat anti-human immunoglobulin G antibody.
23. The method as claimed in Claim 22, wherein said labeling effector is an enzyme labeling effector, said enzyme being selected from the group consisting of peroxidase, chymotrypsinogen, procarboxypeptidase, glyce-roaldehyde-3-phosphoric acid dehydrogenase, amylase, phosphorylase, D-Nase and P-Nase.
24. The method as claimed in Claim 22, wherein said labeling effector is a fluorescence labeling effector, said fluorescence being selected from the group consisting of fluorescence isothiocyanate, tetramethylrhodamine is thiocyanate, substituted rhodamine isothiocyanate, rhoda-mine B isothiocyanate, and dichlorotriazine fluorescence.
25. The method as claimed in Claim 23, wherein said labeling effector is peroxidase-labeled Protein A.
26. A method for assaying an antibody to adult T-cell leukemia associated antigen according to fluorescence immunoassay or enzyme immunoassay comprising reacting an insolubilized antigen having an adult T-cell leukemia associated antigen consisting of soluble cytoplasmic protein of an adult T-cell leukemia cell immobilized on an insoluble support consisting of polystyrene beads, with a test sample seram and determining the resulting antigen-antibody complex.
27. A method as claimed in claim 26 further comprising reacting said antigen-antibody complex with a labeling effector capable of specifically binding an anti-body.
28. A method as claimed in claim 27 wherein said effector is peroxidase-labeled Protein A.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP71080/82 | 1982-04-26 | ||
| JP57071080A JPS58187861A (en) | 1982-04-26 | 1982-04-26 | Assay of antibody related to adult type t leukemia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1197776A true CA1197776A (en) | 1985-12-10 |
Family
ID=13450182
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000426688A Expired CA1197776A (en) | 1982-04-26 | 1983-04-26 | Adult t-cell leukemia antigens, method for their preparation and for assaying antibodies thereto |
Country Status (13)
| Country | Link |
|---|---|
| JP (1) | JPS58187861A (en) |
| BE (1) | BE896571A (en) |
| CA (1) | CA1197776A (en) |
| CH (1) | CH652033A5 (en) |
| DE (1) | DE3315081A1 (en) |
| DK (1) | DK181883A (en) |
| ES (1) | ES8501530A1 (en) |
| FR (1) | FR2525475B1 (en) |
| GB (1) | GB2122343B (en) |
| IT (1) | IT1197633B (en) |
| NL (1) | NL8301464A (en) |
| PH (1) | PH19194A (en) |
| SE (1) | SE8302329L (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5962527A (en) * | 1982-09-30 | 1984-04-10 | Eisai Co Ltd | Preparation of antigen relating to adult t-cell leukemia |
| JPS59151885A (en) * | 1983-02-18 | 1984-08-30 | Eisai Co Ltd | Cell strain related to leukemia of t cell of adult |
| US4743678A (en) * | 1983-04-27 | 1988-05-10 | President And Fellows Of Harvard College | Method and products for detection of human T cell leukemia virus |
| JPS6044870A (en) * | 1983-08-22 | 1985-03-11 | Fujirebio Inc | Detecting reagent for leucocythemia virus antibody of t-cell of adult |
| JPS60249058A (en) * | 1984-05-25 | 1985-12-09 | Eisai Co Ltd | Method and reagent for measuring atl virus antibody |
| US5643714A (en) * | 1986-12-31 | 1997-07-01 | Genelabs Technologies, Inc. | Method and assay for HTLV |
| US5614366A (en) * | 1986-12-31 | 1997-03-25 | Genelabs Technologies, Inc. | HTLV-I peptide antigens and kit |
| JP2525054B2 (en) * | 1989-08-01 | 1996-08-14 | 株式会社トクヤマ | Adult T-cell leukemia virus infection diagnostic agent |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1193378A (en) * | 1967-04-11 | 1970-05-28 | Rand Dev Corp | Cancer Antigen Complexes |
| GB2016687B (en) * | 1978-03-20 | 1982-09-08 | Abbott Lab | Sugar coated reagents for solid phase immunoassay |
| FR2435715A1 (en) * | 1979-01-31 | 1980-04-04 | Sanyo Chemical Ind Ltd | Solid-phase immunological conjugates - comprising active substance coupled to frosted glass |
| CA1148859A (en) * | 1979-06-14 | 1983-06-28 | Lacy R. Overby | Simultaneous assay of two hepatitis viruses using a solid phase |
| DE3005495C2 (en) * | 1980-02-14 | 1983-03-31 | Institut Pasteur, 75724 Paris | Production of fragments of viruses with lipid envelopes and pharmaceutical preparations containing them |
-
1982
- 1982-04-26 JP JP57071080A patent/JPS58187861A/en active Granted
-
1983
- 1983-04-25 DK DK181883A patent/DK181883A/en not_active Application Discontinuation
- 1983-04-25 SE SE8302329A patent/SE8302329L/en not_active Application Discontinuation
- 1983-04-26 ES ES522186A patent/ES8501530A1/en not_active Expired
- 1983-04-26 CA CA000426688A patent/CA1197776A/en not_active Expired
- 1983-04-26 CH CH2219/83A patent/CH652033A5/en not_active IP Right Cessation
- 1983-04-26 PH PH28819A patent/PH19194A/en unknown
- 1983-04-26 NL NL8301464A patent/NL8301464A/en not_active Application Discontinuation
- 1983-04-26 BE BE0/210632A patent/BE896571A/en not_active IP Right Cessation
- 1983-04-26 DE DE19833315081 patent/DE3315081A1/en not_active Ceased
- 1983-04-26 IT IT48154/83A patent/IT1197633B/en active
- 1983-04-26 FR FR8306843A patent/FR2525475B1/en not_active Expired
- 1983-04-26 GB GB08311373A patent/GB2122343B/en not_active Expired
Also Published As
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|---|---|
| ES522186A0 (en) | 1984-12-01 |
| NL8301464A (en) | 1983-11-16 |
| IT1197633B (en) | 1988-12-06 |
| SE8302329L (en) | 1983-10-27 |
| ES8501530A1 (en) | 1984-12-01 |
| FR2525475B1 (en) | 1985-10-31 |
| PH19194A (en) | 1986-01-28 |
| CH652033A5 (en) | 1985-10-31 |
| GB8311373D0 (en) | 1983-06-02 |
| JPH0143908B2 (en) | 1989-09-25 |
| IT8348154A0 (en) | 1983-04-26 |
| BE896571A (en) | 1983-10-26 |
| SE8302329D0 (en) | 1983-04-25 |
| GB2122343A (en) | 1984-01-11 |
| JPS58187861A (en) | 1983-11-02 |
| DE3315081A1 (en) | 1983-11-03 |
| GB2122343B (en) | 1985-09-04 |
| FR2525475A1 (en) | 1983-10-28 |
| DK181883A (en) | 1983-10-27 |
| DK181883D0 (en) | 1983-04-25 |
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