Note: Descriptions are shown in the official language in which they were submitted.
s~
BA ~ OUND OF THE INVENTION
1. Field of the Invention:
This invention relates to a method for determining
human prolyl 4-hydroxylase, whlch is useful for the
straightforward diagnosis of hepatic diseases.
More particularly, this invention relates to a
method for determining human prolyl 4-hydroxylase by
enzyme immunoassay according to the sandwich technique
using a specific monoclonal antibody and/or a polyclonal
antibody.
2. Description of the Prior Art:
Among methods known hitherto for determining human
prolyl 4-hydroxylase (referred to hereinafter simply as
hPH) in human blood is included a method wherein 4-L-
proline in protocollagen
,.
"''t '~
~ , ,, ~ . ... .
' ', .. :i ' :~
57~4
labeled with H is used as a substrate and the resultant H
labeled water is cap-tured by vacuum distilla-tion and measured
Eor its radioac-tivity (Hutton et al., Anal. Biochem., 16,
l 384-394, 1966). Other known methods involve -the use of 14C-
proline labeled protocollagen as a substrate followed by themeasurement of radioactivity from the resultant 4-hydroxylated
C-prollne (Juva et al., Anal. Biochem. 15, 77-83, 1966);
th use of (pro-pro-gyl)5 or (pro-pro gyl)10
followed by the capture and measurement of CO2 released from
2-oxo(1-14c)-glutaric acid (Berg et al., J. Biol. Chem., 248,
1175-1182, 1973). Any of these methods, however, has disad-
vantages of requiring complicate, tremendous operations and
time-consuming measurements. Furthermore, a simple measurement
of hPH activity in blood does not re~lect the txue hPH level,
because most of the hPH is present in blood in an enzymologically
inactivated state.
Under the circumstances described above, it was ~uite
impossible to measure the quantity of hPH precisely in `a simple
manner by way of an enzymatic activity-measuring method.
Consequently, there is a great demand for developing a new
method for effectively and precisely determining hPH in a si~ple
manner in place of the conventional methods accompanIed with
various disadvantages, especially in the field of diagnosis of
hepatic diseases.
BRIEF SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to
provide a method for the enzyme immunoassay of hPH applicable~
to the diagnosis of hepatic diseases.
It is another object of the present invention to provide
' ' '~
57 ~'~
a method for determining hPH by enzyme immunoassay
according to the sandwich technique u6ing a specific
monoclonal and/or a polyclonal antibody to hPH without
accompanying drawbacks as seen in the conventional
methods~
It is still another object of the present invention
to provide a method for determining hP~I by enzyme
immunoassay according to the sandwich technique with a
smaller amount of samples in a simple operation.
Other objects, features and advantages of the
present invention will become apparent as the
clescription proceeds.
According to the invention there is provided a
method for determining human prolyl 4-hydroxylase by
enzyme immunoassay according to the sandwich technique
using a monoclonal antibody or a polyclonal antibody
each to human prolyl 4-hydroxylase, or a mixture
thereof, characterized in that either of the monoclonal
antibody and the polyclonal antibody is used as an
antibody to be`coated onto a solid phase and either of
the monoclonal antibody and the polyclonal antibody is
used as an antibody to be labeled with an enzyme.
B _ F EXPLANATION OF THE DRAWINGS:
Figs. 1 and 2 show hPH standard curves; Fig. 3
shows a graph illustrating hP~I levels in sera from
healthy subjects; Fig. 4 shows an hPH st~ndard curve on
~, one step
: ~ . ' ' :
: ~, . . -~: .,
.: - `'' .
.,
s~
- 3a -
assay; Fig. S shows a graph illustrating an elution
profile on gel filtration of human sera as well as hPH
levels therein; Fig. 6 shows a pattern obtained when hPH
fractions were electroporesed and then transferred to
nitrocellulose sheet.
DETAILED DESCRIPTION OF THE INVENTION
_____________________________________
As a result of extensive researches made by the
present inventors for developing a simple method for
determining hPH in a more straightforward and specific
10manner, it has now been found that a precise and rapid
determination of hPH can be carried out with a smaller
amount of samples by a method utilizing an enzyme
immunoassay ~EIA) according to the sandwich technique
using a monoclonal and/or polyclonal antibody to hPH.
15In accordance with the present invention, there is
provided a method for determining hPH by enzyme immunoassay
:. :
:
~, .
. : :: .
:~Z~57'~
according to the sandwich technique using a monoclonal antibody
and/or a polyclonal antibody each to hP~, characterized in that
either of the monoclonal antibody and the polyclonal antibody
is used as an antibody to be coated onto a solid phase and
either of the monoclonal antibody and the polyclonal antibody
is used as an antibody to be labeled with an enzyme.
The specific monoclonal antibody employed in the method
of this invention is an anti-human prolyl 4-hydroxylase antibody
of IgG, IgA and IgM classes, which is obtained by immuning
an anima] such as a mouse with hPH to form a hybridoma from
anti-human prolyl 4-hydroxylase antibody-producing cells of the
animal and myeloma cells, cloning the hybridoma and thereafter
selecting and cultivating clones capable of producing anti-human
pxolyl 4-hydroxylase antibody having reactivity with the hPH.
This monoclonal antibody has an immunocross reactivity with any
one of the antigenic determinants existing in hPH. This
monoclonal antibody can be purified, if neces~ary, by fractiona-
tion with a sulfate such as ammonium sulfate followed by column
chromatography with DEAE-Sephacel (Pharmacia Fine Chemicalsl
equilibrated with a buffer of a particular pH value.
The polyclonal antibody employed in the method of this
invention is obtained by immuning an ani~lal such as a rabbit
with hPH, taking blood from the immuned animal and purifying
the resultant anti-serum.
Employed as the antibody to be labeled with an enzy~e in
the method of this invention is an IgG fraction obtainable by
the fractionation of a material containing antibodies with
ammonium sulfate or sodium sulfate and the subsequent purifica-
tion on a DEAE-cellulose column. In case of the polyclonal
antibody, it is preferred to carry out ~urther purification on
~rade Mark
:: . . , ....... . :
. .
..
.... ,, :,
'7~4
a ~epharose 4B af:Ei.nity column because this would enhance the
specificity. It is also possible to use F(ab')2 obtainable by
digestion with pepsin or i~s reduced product Fab'. Thus,
the present inventlon includes such an embodiment wherein the
monoclonal and polyclonal antibodies used in the method of this
invention may be their specific binding sit.es F(ab'~2 of Fab'
as such. Besides this, galactocidase or the like technically
available material may be used for this purpose.
The solid phase to be coated with the monoclonal or poly-
clonal antibody should normally be inert to all the substancesused for the antigen-antibody reaction including a liquid
vehicle and is selected from awide variety of inorganic and
organic inert carrier materials such as glass, ceramics and
resinous materials in the form of a plate or sphere. Such solid
phase should be homogeneous in quality and identical in size,
or otherwise, the quantity of the monoclonal or polyclonal
antibody coated on the individual solid phase fluctuates, thus
resulting in an inaccurate result of measurements. Because of
easiness in processing, organic resinous materials such as
polystyrene, polyvinyl resin, polyamide resin in the form of
a plate or sphere is preferable, with polystyrene bal.~s and
polyvinyl chloride plates being most preferable.
A various kinds of buffer solution can be used for the
immunoassay of the present invention to provide a definite pH
value desirable in the system. A buffer substance used or
: this purpose is selected from various known compounds having
a bufering action, according to the conditions required in the
system. Preferable examples of the bu~fer substances include
¦ phosphates, tris-HCl, acetates and amino acids. These sub-
stances are used with an acid or sodium chloride at a concent- .
_ 5 _
.. . .
- .., . , : -.-.
,
~Z~5'7'~ 1
ration desired in the sys-tem. The operations .~or the immuni-
~zation, chromatoyraphy, equilibratlon, fractionation, fluorim~
etry and spectropho-tometry in the method of this invention can
l be carried out according -to the methods known per se for these
5 ¦ purposes.
The method of this invention is carried out especially
advantageously by using the monoclonal antibody to hPH as the
antibody to be coated onto the solid phase and/or the antibody
to be labeled with an enzyme.
Our recent immunological tests have revealed that a
significant increase in the hPH level is observed in tissue
or blood of patients suffering from liver fibrosis caused by
hepatic diseases such as chronic hepatitis, liver cirrhosis
and alcoholic hepatic disorders.
As will be shown in Table 5 below, the hPH levels in sera
~rom patients with liver cirrhosis as measured in accordance
with the method this invention are significantly higher than
those of sera from healthy subjects. ~ccording to the present
invention, the measurement of blood hP~ levels in a simple
manner enables foreknowing hepatic diseases, especially liver
fibrosis, without relying on biopsy which is burdensome on
patients. We have confirmed that fibrosis of hepatic tlssues
cannot be determined by the conventional liver function tests
relying on measurement of the activity of GOT (glutamate- :
oxaloacetate transaminase), GPT t~utamate-pyruvate transaminase),
LDEI (lactate dehydrogenase), y-GTP ty-gIutamyl transpeptidase),
etc. Thus, the present invention is very useful in the field
of diagnosis of hepatic diseases since detection o~ diseases
of this type at an early stage can be expected by the measure-
ment of blood hPH levels according to the method of this inven-
6 -
:. : .
. ' ., . ~ ` , -
~Z~
Il tion and since the cliagllosis oE fibrosis of hepa-tic tissues
can be made by the method of this invention capable of measuring
hPEI. In addi-tion, the method i-tselE of this invention is simp]e
as compared with the conventional methods and a result obtained
5 j~ by the method of this invention is exact and trustworthy.
Thus, the method of this invention is economically advantageous
¦ over the conventional methods.
I The present invention will now be illustrated in more
¦ detail by way of the following examples, but i-t is to be
10 I construed that the scope of this invention is not limited by
¦I these specific examples. Among these examples, Examples 1 and
2 illustrate processes for the preparation of the monoclonal
Il antibody and the polyclonal antibody respectively, used in the
¦¦ method of this invention and Examples 3-5 illustrate the method
15 1 for determining human prolyl 4-hydroxylase by enzyme
immunoassay according to the present invention.
.
. ,:
lL;2~ 7'~
Example 1
Preparation of mouse anti-hPFI monoclonal antibody
(a) Preparation of antigen-hPH (EC 1.1~.11.2):
¦ Using human placenta as a material, hP~I was puri~ied
by affinity chromatography on poly-L-proline
coupled ~epharose 4B column according to a method
of Tuderman et al. described in Eur. J. Biochem. 52, 9-16
(1975), and was then purified with a Bio-Gel A-l.Sm (Bio-Rad)
column. The hPH preparation obtained was examined for
its purity by electrophoresis with the sodium dodecylsulfate-
polyacrylamide gel (SDS-PAGE) according to a method of Baum
et al. described in J. Virol. 10, 211-219 (1972) whereupon
the purity was about 90%.
(b) Preparation of antibody-producing cells:
Three Balb/C female mice of 8 weeks old were subjected to
first immunization with the hP~i purified in (a~ above in
a complete Freund adjuvant. 50 ~g of hpH as a 0.5 ml
solution was intraperitoneally administered to each mouse.
Further, the mice were subjected to booster immunization
with hPH in the same amount dissolved in physiological saline
on the 30th and 60th days. As a final immunization, the mice
were subjected to subsidiary immunization on the 90th day by
intravenous administration (50 ~g/100 ~1 physiological saline).
After 3 days~ the mice were killed to exti~pate their spleens
and the splenocytes were harvested.
(c) Cell fusion:
The following materials and methods were employed:
RPMI 1640 culture medium was prepared by adding sodium
bicarbonate (12 mM), sodium pyruvate ~1 mM), L-glutamine
(2 mM), penicillin G potassium (50 u/ml), streptomycin sulfate
: . ~
ll ~26~i7~
(50 ~g/ml) ~nd amlkacin sulfate (100 ~g/ml) to RPMI No. 1640
(Difco Laboratories), adjusting pH to 7.2 with dry ice and
sterilizing the mixture by filtration with a 0.2 ~m Toyo membrane
filter.
NS-l culture medium was prepared by adding a fetal bovine
serum (Granite Diagnostic, Inc.) which had been sterilized by
filtra-tion to the RPMI-1640 culture medium above at a concentra-
tion of 15~ (v/v).
HAT selection medium was the NS-l culture medium further
containing hypoxanthine (100 ~M), aminopterine (0.4 ~M) and
thymidine (16 ~M).
HT culture medium was of the same composition as the HAT
selection medium except that the aminopterine had been removed
therefrom. PEG 4000 solution was prepared by dissolving
polyethylene glycol 4000 (PEG 4000, Merck ~ Co., Inc.) in the
RPMI 1640 culture medium so that a 50% (w/w) non-serum solution
was obtained.
The fusion with 8-azaguanine-resistant myeloma cell lines r
NS-l (P3-NS 1-1) was carried out by somewhat modifying the
method of Oi et al. described in Selected Method in,Cellular
Immunology (ed. B.B. Mishell and S.M. Shiigi), W.H. Freeman
and Company (1980), 351-372. 1.5x108 karyo-splenocytes
(cell viability: 95%) were fused w~th 2.8x107 cells of NS-l
myeloma cells (cell viability: 95%). The karyo-spelenocytes
and myeloma cells were separately washed with the RPMI-1640
culture medium described above. They were suspended in the
same culture medium whereby they were mixed in the ratio
he~einbeEore described for fusion. Using a 50 ml conical test
tube made of styrene resin (Corning Glass Works), the mixture
.
_~_
: .
; , ,
. .
,~
. ., ~ : ~
'' ': ,
:~Z~ 14
in 40 ml of the RPMI-1640 culture medium was centrifuged for
10 minutes at 400xg to remove the supernatant completely by
suction. To the precipitated cells was added dropwise over
one minute under gentle agitation 1 ml of the PEG 4000 solution
warmed at 37C. Further gentle agitation was carried out for one
minute to resuspend the cells for dispersion. Next, 1 ml of
the RPMI-1640 culture medium warmed at 37C was added dropwise
over one minute. After repeating this operation once, 7 ml
of the same culture medium was added dropwise over 2-3 minutes
under continuous agitation to effect dispersion of the cells.
This mixture was subjected to centrifugal separation for 10
minutes at 400 xg and the supernatant was completely removed
by suction. To the precipitated cells was added immediately
10 ml of the NS-l culture medium warmed at 37C, and large
lumps of cells were carefully pipetted with a 10 ml pipette
for dispersion. Further, 20 ml of the same culture medium
was added to dilute the dispersion, and it was distributed in
l a 96-well microplate (Corning Glass Works) made of polystyrene
2 so that 5.9 x 10 cells~0.1 ml of the culture medium may
0 exist in each well. As a preliminary treatment of the 96-
well microplate to be used, 0.2 ml o~ the NS-l culture medium
was addéd thereto, and the microplate was warmed overnight
in a carbon dioxide incubator (37~C~ and sucked to re ve
the culture medium just before use. The microplates where the
cell fusion has been finished were incubated at a temperature
of 37C and a humidity of 100~ in 7% carbon dioxidej~3~ air.
(d) Selective culture of the hybridoma in the selection
mediwn:
On the first day of incubatian, 2 drops (about 0.1 ml) of
the XAT selection medium were added with a Pasteur pipette.
,. ' : , .
. , ,
.: , .
-. :
On the 2nd, 3rd, 5th, 8th and 11th days, a half of the culture
medium (0.1 ml) was replaced by a fresh HAT selection medium.
On the 14th day, the cultu~e medium was repl~ced b~ the HT
¦ culture medium and the same operat~on was repeated every 3-4
days. The growth of a satisEactory hybridoma (the fusion rate:
83~) was observed usually in 2-3 weeks. All of the wells where
the hybridoma has grown, were checked for positivity according
to a solid phase-antibody binding test (ELISA) described in
the following item te). 20 cells/288 wells were detected
positive. Each positive cell (20 cells/288 wells) detected
and 1 ml of an HT culture medium containing 10 mou,se thymocytes
as a feeder were added to a 24-well plate (Corning Glass Works)
made of polystyrene whereby the whole contents of the 20 positi~e
hybridomas detected were transferred. They were incubated in
the same manner as in (c) above at 37C for about one week in
, the presence of 7% carbon dioxide. During the incubation, 0.5
ml of the super~atant in each well were replaced once or twice
by 0.5 ml of a fresh HT culture medium. At the time the
hybridoma had well grown, its positivity was reconfirmed by
ELISA and each hybridoma was subjected to cloning according
to the limiting dilution method described in the item ~f) below.
The residual solution after the,use for the cloning was trans-
ferred to a 25 cm tissue culture flask (Corning Gla~s Works)
made of polystyrene to prepare a sample for storage under
freezing.
(e) Screening o~ hybridoma capable of secreting the
anti-hPH antibody according to the solid phase-
antibody binding test (ELISA):
A method somewhat modifying a method of Rennard et al.
described in Anal. Biochem. 104, 205-219 (1980) was employed.
..
' - 11 -
....
,
.
'
- 12 1 ~ ~ 5~7L~
This method is suitable ~or the detection of antibodies rom
hybridoma. A 96-well micro~itration plate (Flow Laboratories,
Inc.) was coated with 0.5-1.0 ~g of hPH and the others were
blocked with 1% bovine serum albumin (BSA). To this was added
a part of the supernatant of the hybridoma-grown well, and
the incubation was carried out for about one hour at room
temperature. A horseradish peroxidase conjugated goat anti-mouse
IgG (TAGO, Inc.) as a secondary antihody was added and further
incubation was carried out for about one hour at room temperature.
Next, hydrogen peroxide and o-phenylene-diamine as a substrate
were added, and the degree of the resultant brown color was
evalua~ed qualitatively with naked eyes or the absorbance at
500 nm was determined with a CORONA*double wave micro-plate
spectrophotometer (MTP-22, Corona Denki Xabushiki Kaisha).
(f) Cloning:
Since there was a possibility of at least 2 kinds of
hybridoma heing grown in each well, cloning was conducted
according to the limiting dilution method to obtain a mono-
clonal antibody-producing hybridoma~ A cloning culture medium
was prepared which contained 107 mouse thymocytes as feeder
per ml of the NS-l culture medium, ana was added to 36, 36 and
24 wells of a 96-well microtitra~ion plate at 5, 1 and 0.5
hybridomas per each welL, respectively. On the 5th and 12th
days, about 0.1 ml of NS-l culture medium was additionally added.
A satisfactory growth of the hybridoma was observed 14~15 days
a~ter the cloning, and ELISA was carried out for the group
where negative colony-forming wells were more than 50%. In
case all hybridoma3 in the tested wells were not positive,
the number of colonies in the antibody-positive wells was checked
land hybridomas from 4-6 wells were selected from the wells
~herein one colony existed and again subjected to cloning.
* ~rade Mark
.
, .
: -
. ::
: '';., ' ' ~''
l~c;S~
Ultimately, 8 clones were obtained.
(g) Culture in vitro and in vivo of the monoclonal antibody:
The resultant clone was incubated in the NS-l culture
medium or the like proper culture medium (in vitro culture),
and a monoclonal antibody could be obtained from the super-
natant of the cultivated medium (the concentration of the
monoclonal antibody protein: 10-100 ~g/ml). In order to obtain
the antibody in a larger amount, on the other hand, Pristane,
a tumor formation-accelerator (2,6,10,14-tetramethylpentadecane,
Aldrich Chemical Company, Inc.), was intraperitoneally
administered to the same animal (Balb/C mouse) as that providing
the thymocytes and the myeloma cells in a dose of 0.5 ml per
mouse. After 1-3 weeks, 1 x 107 cells of hybridoma are also
l intraperitoneally administered whereby an ascites having a
1 concentration of 4-7 mg protein/ml of the monoclonal antibody
can be obtained in vivo after 1-2 weeks.
(h) The isotype of heavy chain and of light chain of the
¦ monoclonal antibody:
Each of the resultant ascites was first bound to a micro-
titration plate coated with hPH in accordance with ELISA
described above. After washing, an isotype-specific rabbit
anti-moase Ig antibody (Zymed Laboratories) was added. After
washing, horseradish peroxidase labeled goat anti-rabbit IgG
(H+L) antibody was added and was then detected with 2,2'-azino-
di(3-ethylbenzthiazoline sulfate-6) as a substrate and hydrogen
peroxide. The results are arranged and shown in Table 1.
Among the investigated antibodies, four antibodies had immuno-
globulin chains rl/~, one antibody r2b/~, two antibodies ~/~
and one antibody ~/~.
Furthermore, each of the resultant monoclonal antibodies
~,
:` ' ` ~ .,~ ,
, - ~ .
- ` : ~
.
.
. ~"
5'7 ~
was checked for cross-reactivity with hP}I subunits by
the western blotting method described by Towbin et al. in
Proc. Natl. Acad. Sci. USA, 76, 4350 4354 (1979)~ E~our
out of the 8 resultant monoclonal antibodies reacted with
~ chain with molecular weight of 64 ~D and the remaining 4
antibodies with 3 chain with molecuLar weight of 60 KD (see ~able 1 ;
for subunits, see Chen-kiang et al., Proc. Natl. Acad. Sci.
USA, 74, 4420~4424, 1977).
(i) Purification of monoclonal antibody:
Each ascites obtained in (g) above was fractionated with
ammonium sulfate (40% saturation) and subjected to colurnn
chromatography with DEAE-Sephacel (Pharmacia Fine Chemicals)
equilibrated with 40 mM phosphate bufer solution, pH 8.0,
containing 0.06 M sodium chloride to obtain the IgG class in an
unabsorbed fraction therefrom. This IgG class was further
¦subjected to gel filtration with a Sephacryl S-300 Superfine
(Pharmacia Fine Chemicals) column equilibrated with 50 mM
phosphate buffer solution, pH 7.4, containing 0.42 M sodium chlo-
ride to separate and remove the fetal bovine serum in the
culture medium and mouse proteins, The IgA and IgM classes
were purified under the same conditions as in the case of
other IgG classes except that they were eluted from a DE~E-Sephacel
column with a gradient from 0.06 M to 1.0 M sodium chloride.
Example 2
Preparation of antiserum and rabbit anti-hPH polyclonal antibody
(a) Immunization:
A female rabbit was subjected to first immunization with the
pHP which was isolated and purified from human placenta in the
same mar.ner as in Example l(a)~ in a complete Freund adjuvant.
A mixture of 200 ~g of hPH and 1 ml of the adjuvant was
', ' ,
- 15 - ~ ~t~5t~ 4
subcutaneously administered to the back in lS positions, after
which 200 ~g of hPH in the complete Freund adjuvant was
subcutaneously administered to the back of the rabbit every
2 weeks over the period of 4 months to effect booster
immunization. After each booster immunization, a blood sample
was taken to check its antiserum for anti-hPH activity in
accordance with the method described by Hutton et al. in
Anal. Biochem. 16, 384-394 (1966). There was exhibited 56%
inhibitory of the activity with 4 ~1 of the antiserum. This
antiserum was judged to be specific to the hPH from the
fact that it formed only one precipitin line when subjected to
Ouchterlony immunodiffusion and immunoelectrophoresis.
(b) Purification of antiserum:
The rahbit antiserum obtained in (a~ above was fractionated
with sodium sulfate tl8% saturation) and then applied to column
chromatography with DEAE-cellulose (DE52, Whatman) equilibrated
with 17.5 mM phosphate buffer solution, pH 6.3, to obtain
an anti-hPH polyclonal antibody in unabsorbed fractions
(purified IgG fractions).
Example 3
Sandwich enzyme immunoassay for hP~
(a) Procedure for the preparation of rabbit anti-hPH
IgG-POD conjugate: ,
Rabbit anti-hPH IgG-POD conjugate was prepared in accordanse
with the mothod described by Ishikawa in J. Immunoassay, 4,
209-327, 1983. The rabbit anti-hPH IgG obtained in Example 2(b)
above was dialyzed against 0.1 M phosphate buffer solution,
2H 6.5. To 0.3-0.5 ml of the rabbit anti-hP~I IgG dialy~ate was
added a 100-fold molar ~uantity of S-acetylmercaptosuccinic
anhydride dissolved in dimethylformamide. The mixture was
* Trade Mark
~ A ,
~' '` ' ' . ' ,. ~ ,, , . ~
" .' ~ ' ~ '
- 16 - ~ '7~
incubated at 30C for 30 minutes. 100 ~1 of 0.1 M Tris-HCl
buffer, pH 7~0, 10 ~1 of 0.l M EDTA solution, pH 6.0, and l00 ~l
of 1 M hydroxylamine solution, pH 7.0 were then added. Thè
mixture was allowed to stand at 30C for 5 minutes and then
subjected to gel filtration with Sephadex G-25 equilibrated
with 0.1 M phosphate buffer solution, pH 6.0, containing 5 mM
EDTA. In this manner there was obtained SH group-labeled
rabbit anti-hPH IgG.
Separately from the procedure described above, horseradish
peroxidase (POD) was labeled with a maleimide. Thus, 6 mg of
POD was dissolved in 0.1 M phosphate buffer solution, pH 7.0,
and a 25-fold molar quantity of N-(~-maleimidocaproyloxy)
succinimide dissolved in dimethylformamide was added to the
solution. The mixture was incubated at 30C for 30 minutes.
It was then subjected to gel filtration with Sephadex G-25
equilibrated with 0.1 M phosphate buffer solution, pH 6.0, to
collect maleimide-labeled POD fractions.
To one mole of the above prepared SH group~labeled IgG
was added about 5 moles of the maleimide-labeled POD prepared
above, and the mixture was allowed to stand at 4C for 20
hours. The mixture was then subjected to gel filtration on
an Ultrogel* AcA 44 (LKB) column equilibrated with 0.1 M phosphate
buffer, pH 6.5, to collect rabbit anti-hP~ IgG-POD conjugate
fractions. BSA and thimerosal were added as a stabilizer and a
preservative at concentration of 0.1% and 0.005~, respectively,
and the fractions were stored at 4C until use.
Also for the mouse anti-h~l~ monoclonal antibodies,
the same treatment as described above was applied to prepare
corresponding monoclonal antibody IgG-POD conjugates.
tb) Procedure for the preparation of rabbit anti-hPH
~ i * Trade Mark
"'', ' ~
,
~2~;5'7~
Fab'~POD conjugate:
ll Rabbit anti-hPH IgG obtainecl in Example 2(b) above was
¦I dialyæed against 0.1 M acetate buffer solution, p~l 4.5, containing¦
~l 0.1 M sodium chloride. The thus purified anti-hPH IgG was
5 1I digested at 37C for 24 hours with pepsin added at 2~ (w/w) to
¦ the antibody. The reaction was terminated with 2 M Tris-Hcl buffe
¦ solution, pH 8.0, and the reaction mixture was subjected to gel
¦ filtration on an Ultrogen AcA 44 column equilibrated with 0.1 M
phosphate buffer, pH 7.0, to collect F(ab')2 fractions. The
I F(ab')2 fractions were then dialyzed against 0.1 M phosphate
¦ buffer solution, pH 6.0, to prepare a 0.5-5 mg/450 ~1 solution.
To this solution was added 50 ~1 of 0.1 M mercaptoethylamine
dissolved in 0.1 M phosphate buffer solution, pH 6.0, containing
l 5 mM ~DTA, and the mixture was incubated at 37~C for 90 minutes.
15 ¦ The mixture was then subjected to gel filtration on an Ultrogel
j AcA 44 column equilibrated with 0.1 M phosphate buffer solution,
I pH 6.0,containing 5 mM EDTA to collect Fab' fractions.
I ~ Since the thus prepared Fab' is labile, maleimide-labeled POD
l prepared in the same manner as in (a) above was added thereto
20 1 within 24 hours in an amount equimolar to that of the Fab ' .
O.1 M phosphate buffer solution, pH 6.0, containing 5 mM EDTA
was further added so that the respective final concentrations
of 50-100 ~M were given. The resultant mixture was allowed to
stand at 4~C for 20 hours or at 30~C for one hour. The ~ree
SH groups were then blocked with N-ethylmaleimide in a 10-fold
molar quanti~y relative to the Fab'. The mixture was subjected
to gel filtration on an Ultrogel AcA 44 column equilibrated
with 0.1 M phosphate buffer solution, p~ 6.5, to collect rabbit
anti-hPH Fab'-POD conjugate ~ractions.
1! ~ ALto for the mouse anti-hPH monoclonal antibodies,
~ - 17 -
'' ' : "' : '
:
-: . ::
. :' : ~ ~ ' ' " ''
., . ~ .
,: ~. ' ' " ' :"' ~' ,,
~2~5~ L~
the same procedure as described above was followed to prepare
corresponding monoclonal antibody Fab'-POD conju~ates.
(c) Procedure for the determination of ratio of specific bindin~:
l Although the POD labeled antibodies prepared in (a)
~ and (b) above can usually be used as such, i.e. without further
¦I purification, it is desirable, where the specific binding
~¦ ratio (us~ully 7-8%) is less than 1%, to further purify by
specific purification which will be referred to below.
100 ng of the rabbit anti-hPH IgG-POD or rabbit
anti-hPH Fab'-POD was first dissolved in 100 ~1 of 10 m~l
phosphate buffer solution, p~E 7.0, containing 0.1 ~ sodium chlori~e
and 0.1% BSA and the solution was loaded onto a hPH-coupled
Sepharose 4B affinity column (100 ~1 gel). The column was
washed with 3 ml to 10 m~l phosphate buffer solution, pH 7.0,
I containing 0.1 M sodium chloride and 0.1~ BSA. The
¦ same operation was applied to a BSA-coupled Sepharose 4B column.
To a 10 ~1 portion of each effluent were added 250 ~1 of 0.5%
p-hydroxyphenyl--acetic acid (PHPA) dissolved in 50 m~E acetate
l buffer solution, pH 5.0, and 50 ~1 of 0.01% aqueous hydrogen
1 peroxide solution, and the mixture was incubated at 30C for
10 minutes. 2.5 ml of 0.1 M glycine-sodium hydroxide buffer
¦ solution, p~E 10.3, was added to stop the reaction and the
¦ relative fluorescence intensity of the reaction mixture was
measured using quinine ( 1 ~g/ml of 0.1 N sulfuric acid) as
a control. The difference of the total POD activity of the
effluent from the BS~- or hPEI-coupled Sepharose 4B column
was used to determine the specific binding ratio.
(d) Procedure for affinity purification:
Anti-hPH I~G-POD or anti-hPE-I Fab'-POD
was applied to a hPH-coupled Sepharose 4~ column (200 ~1
- 18 -
`: ' .~.'` '
~1 :azl~s~7l~
~¦gel) equilibrated with 10 mM phosphate buffer solution, pH 7.0,
containing 0.1 M sodium chloride and o.l % BS~, and then the
column was wash~dwith 5 ml oE the same buffer
as described above. Specific antibody was eluted with 500 ~1
of 0.1 M glycine-~lCl buffer solution, p~ 2.5 and the effluent
was immediately neutralized with 1.0 ml of 0.5 M
Tris-HCl buffer solution, pH 8Ø The effluent
was concentrated to 0.1-0.3 ml and the concentrate was subjected
to gel filtration on an Ultrogel AcA 44 column which was previously
equilibrated with 0.1 M sodium phosphate buffer solution, pH 6.5,
containing 0.1% BSA to collect antibody-POD conjugate fractions.
After addition of thimerosal, the fractions were stored at 4C
¦until use.
¦ With the monoclonal antibodies, which were high in the
specific binding ratio, there was no need to apply this affinity
purification.
(e) Procedure for the preparation of antibody~coated
polystyrene ball:
The mouse anti-hPH monoclonal antibodies and the rabbit anti-
hPH polyclonal antibody obtained in Examplesl(i) and 2tb),
respectively,were individually dissolved in 0~1M phospha-te
buffer solution, pH 7.5, containing 0.1% sodium azide at a
concentration of 0.1 mg/ml. Polystyrene balls (Precision
Plastic Ball) were immersed at 4C for 24 hours in each
of these antibody solutions to coat the polystyrene balls
with the respective antibodies. After withdrawing of the free
antibody immersion solution, the polystyrene balls were washed,
before use, 5 times with "buffer ~" (10 mM phosphate buffer,
pH 7.0, containing 0.1~ BSA, 0.1 M sodium chloride and 0.1%
sodium azide). Where allowed to stand at 4C for one week or more
.. . .
'' ' . ' ~
' '
l~S'7'14
they were washed again, upon use, three times with
buffer A.
(f) Assay procedure:
l The polystyrene balls prepared in (e) above were subject-.
¦ ed to assay by a fluorimetry method (using balls of 3.2
mm in diameter) and by a colorimetry(using balls
of 6.5 mm in diameter).
1) Fluorimetry: .
Ten test tubes(8 ~m in inner diameter and 75 mm in
length) in duplicate were prepared. 150 ~1 portions of
the standard hPH preparation purified in Example l(a) and
diluted to concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1, 3,
10, 30 and 100 ng/150 ~1 were added individually to the tubes.
Ten~l of human serum and also 140 ~1 of buffer ~
were added to a test tube for sample and mixed well
with one another. One antibody- coated. ball prepared in
Example 3(e) was lightly picked up w.ith a pincette, fred of
the adhering liquid by absorbing it with filter paper and put
in each test tube. Each test tube was incubated at 37C for 1-4
hours with continuous shaking (~irst reaction; the tesk tube may,
after this reaction, be allowed to stand at 4C one day). The
reaction solution in each test tube was sucked off, 2-3 ml of a ~
washing solution (10 mM phosphate buffer, pH 7.0 containing 0.1 M
sodium chloride)was added twice to wash the contents and
the washes were removed by suction. Using o~her test tubes,
the enzyme-labeled antibodies prepared in Examples 3(a)
and (b), respectively, were diluted with 10 mM phosphate buffer
solution, p~l 7.0, containing 0.1~ BSA and 0.1 M sodium chloride to
a concentration of 100 ng/150 ~1. The washed polystyrene balls
~0 described above were transferred to these test tubes (one ball/
- 20 -
. .
.
~Z~5~7~
j tube). It is to be noted -tha-t, in the case of the specifically
purified enzyme-labeled antibody in Example 3(d) above, a
dilution of 10 ng/150 ~1 was used. Each testtubewas incubated
at 20C for 3-4 hours wi-th continuous shaking (second reaction)
5 ~l and the unreacted enzyme-labeled antibody solution in each test
¦I tube was sucked off and the residue was washed twice with 2-3 ml
of a washing solution. To other test tubes was added 100 ~1 of
¦ 0.1 M phosphate buffer solution, pH 7.0 having dissolved therein
¦ 0.6~ 3-(p-hydroxyphenyl)propionic acid (HPPA) as a PO~ substrate.
10 ¦¦ The washed polystyrene balls were transferred to these test
tubes, one ball for each test tube. Two test tubes containing
the POD substrate, HPPA alone were prepared in this reaction
step as blank and subsequent operations were also applied thereto.
To each of the substrate-placed test tubes was added 50 ~1 of
0.015% aqueous hydrogen peroxide solution, and the tubes were
incubated at 30C for one hour (enzymatic reaction) with
j continuous shaking. 2.5 ml of 0.1 M glycine-sodium hydroxide
buffer solution, pH 10.3, was added to stop the reaction. The
relative fluorescence intensity was measured with a Hitachi
spectrofluorometer (Model 204) using quinine (1 ~g~ml of 0.1 N
. sulfuric acid~ as a control~ Using log-log graph paper with
the abscissa indicating the amount in ng of the hPH preparation
and the ordinate indicating the relative fluorescence intensity,
: each measured value for relative fluorescence intensity
m~nus blank value was plotted to draw a standard curve. The
amount of hPH per ml of a sample was calculated by multiplying
hy 100 the reading from the standard curve o the hPH (ng)
corresponding to the relative intensity obtained with 10 ~1 of
the sample.
2~ Colorimetry:
.-' ,. , :'
, . : ,.-
~ ;S7,~9~
en -test tubes (9.5 mm in inner diameter and 105 mm in
length) in duplicate were prepared. 300 ~1 portions of the hPH
preparation purified in Example l(a) alld dilu-ted to concentration
~ of 0, 0.01,0.03, 0.1, 0.3, 1, 3, 10, 30 and 100ng/300 ~1 was
5 ~ added individually to the tubes. 10 ~1 of human serum and also
290 ~1 of the buffer ~ were added to a test tube for sample and
mixed well with one another. One antibody-coated ball prepared
in Example 3(e) was lightly picked up with a pincette, fred of
~ the adhering liquid by absorbing it wi-th filter paper and put
10 1l in each test tube. Each test tuhe was incubated at 37C for
! 1-4 hours with continuous shaking (or at 30C for 1-2 hours)
1~ (first reaction). The reaction solution in each test tube was
'i sucked off, and the residue was washed twice with 2-3 ml portions
~ of the washing solution added. The washed polystyrene balls were
transferred to other test tubes each con-taining 300 ~1 of a
solution containing 10 or 100 ng of one of the enzyme-labeled
antibodies prepared in Examples 3(a) and (b) above (one ball/
tube). All these test tubes were incubated at 20C for 3-4 hours
¦ (or at 30C for 1-2 hours) with continuous shaking (second
reaction). The unreacted enæyme-labeled antibody solution in
each test tube was sucked off and the residue was washed twice
with 2-3 ml of the washing solution~ To separate test tubes
was added 300 ~1 of 0.1 M acetate buffer solution, pH 5.5,
having dissolved therein 0.0134% 3,3',5,5'-tetramethylbenzine
(TMBZj. The washed polystyrene balls were transferred to these
test tubes, ~one ball/tube~. Two test tubes containing the
POD substrate TMBZ alone were prepared in this reaction step
as blanks and subsequent operations were also applied thereto.
To each o~ the suhstrate-placed test tubes was added 100 ~1
o~ 0.01% aqueous hydrogen peroxide solution, and the tubes were
,
: : , : .
.
-.,,~ -` . ' .
. .: .
~ . .
~Z~j5'~
incuba-ted a-t 30C for 45 minutes with continuous shaking
(enzymatic reaction~. 600 ~Jl of 1.33 N sulfuric acid was then
added to stop -the reaction. The absorbance at 450 nm was
measured with a Shimadzu double beam spectrophotometer (model
UV-150-02) usi.ng water as a control, and absorbance values for
samples minus those for blank were obtained. Calculation of
the amount of hPH per ml of the sample was performed in the
same manner as in the fluorimetry described in 1) above.
¦ (g) Screening of optimal pair of antibodies for solid phases
10 ¦ and conjugates: ¦
Before carrying out assay of hPH by the sandwich method
described above, the following screening test was effected to
I¦ determine optimal combinations o~ antibodies as solid phase and
il those as conjugate. The eight monoclonal antibodies and rabbit
anti-hPH polyclonal antibody obtained in Examples 1 and 2,
respectively, were checked for optimal pair us.ing the standard
hPH preparation purified in Example l(a). The results are
shown in Table 2. As for immunoglobulin (Ig)-POD conjugate
¦ (enzyme-labeled antibody), pairs of 2-5G8 / 3-2B12, 2-lC2 /
20 ¦ 3-2B12, 2-6G9 / 3-2B12 and 2-lC2 / rabbit anti-hPH IgG (all
: expressed as solid phase conjugate) exhibited high ratios ~S/N)
of specific binding (S) to non-specific binding (N). In the
case of Fab'-POD conjugate, high S/N ratios were exhLbited with
a pair of 3-2B : 12 / rabbit anti~hPH IgG in addition to the
pairs described above. On the basis of these results, standard
curves were made with the following solid phase / conjugate:
:~ pairs of 2-5G8 / monoclonal antibody IgG(3-2B12~; 3-2B12 /
rabbit anti-hPH Fab' ; and rabbit anti-hPH IgG / rabbit anti-
hPH Fab' (see Figs. 1 and 2). Fig. 1 shows an hPH standard
curve obtained by the Eluorimetry wherein the assay was carried
I 23 - !
. - . , . ;
~2~S~
out at 37C for one hour Eor the first reaction; at 20C for
3 hours for the second reac-tion; and at 30C for one hour for
the third reaction with continuous shaking. In Fig. 1, (-~-)
~ is for the solid phase being rabbit anti-hPH Ic3G, and (-o-)
5 ~ for the solid phase being monocLonal antibody (3-2B12). In
both case, rabbit anti-hPH Fab'-POD was used as the conjugate.
(-~-) is for the solid phase being monoclonal antibody (2-5G8)
and the conjugate being monoclonal antibody IgG (3-2B12!-POD.
Fig. 2 shows a hPH standard curve obtained by the color-
imetry wherein the assay was carried out at 37C for one hour
for the first reaction; at 20C for 3 hours for the second
reaction; and at 30C for 45 minutes for the third reaction
with continuous shaking. In Fig. 2, (-o-) is for the solid
phase being rabbit anti-hPH IgG, and (-o-) for the solid phase
being monoclonal antibody (3-2B12). In both case rabbit
anti-hPH Fab' POD was used as the conjugate. In both o~ the
fluorimetry and the col-orimetry~ Linearity was obtained in the
range of 0.1-100 ng of hPH per test tube, and the sensitiyity
was 0.1 ng. Somewhat better sensitivity was observed to be
obtained with a pair of both rabbit anti-hPH polyclonal anti--
bodies, for solid phase and conjugate, than with a pair of
monoclonal antibody / polyclonal antibody or monoclonai antibody/
~monoclorlal antLbody.
Using 10 ~1 each o~ human serum samples from healthy
subjec~ts, screening of optimal pair of monoclonal antibodies
for solid phase and/or conjugate was carried ou-t ~see Table 3).
As clearly shown in Table 3, specific binding was extremely
low in any of the pairs. On the other hand, where 10 ~1 each
of human serum samples from healthy subjects was likewise used
With the both rabbit anti-hPH IgG as solid phase and as conjugate~
.
~-
1~65~
"
the ~luorescence intensity was Eound to increase wi-th increasing
¦l amounts of the serum. Thus, Fig. 3 depicts hPH levels in 0 50
~ l of serum from healthy subject measured by the fluorimetry
j~ wherein assay was carried out at 37~C ~or one hour for the
5 l~ first reac-tion; at 20C for 4 hours for the second reaction;
and at 30C for one hour for the third reaction with continuous
i shaking. In Fig. 3, the solid line ( - ) is for the solid phase
being rabbit anti-hPH IgG and the conjugate being rabbit anti-
hPH IgG-POD, and the dotted line (----) is for the solid phase
10 ¦ being monoclonal antibody (2-5G8) and the conjugate being
¦, monoclonal antibody IgG (3-2B12)-POD. On the other hand, no
~ specific binding was observed when monoclonal antibody (2-5G8)
¦¦ was used as solid phase and monoclonal antibody IgG (3-2B12)-POD
Il as conjugate. Thus these results show that hPEI cannot be
15 ¦I quantitatively determined with pairs of the monoclonal antibodies
Il alone. Table 4 shows the results obtained when the monoclonal
¦¦ antibodies or rabbit anti-hPH antibody were used individually
¦ as solid phase and the rabbit anti-hPH IgG or Fab'-POD as a
l conjugate. Satisfactory ratios (S/N) of specific binding (S)
20 to non-specific binding (N) resulted when the conjugate was
rabbit anti-hPH IgG-POD and the so~id phase was clones, 3-2B12,
3-6H5, 2-6G9, 2-7F8 or rabbit anti-hPH IgG. Satisfactory
results were also obtained where the conjugate was rabbit
anti-hPH Fab~-POD and the solid phase antibody was 3-2B12,
3-4H2 or rabbit anti-hPH IgG. Less`non-specific binding was
observed with Fab'-POD than with IgG-POD as conjugate.
(h) Determination of hPH levels in sera:
i Based on the screenin~ of optimal pair of antibodies for
use AS solid phase and conjugate antibodies in tg) above,
monoclonal antibody IgG(3-2B12) or rabbit anti-hP~I antibody IgG
' :; ` '
:, ~` .
, ,...... ~ :
3L~ti5'7~
was used as solid phase and rabbit anti-hPH Fab'-POD was used
as conjugate to determine hPH levels in sera from healthy
subjects and patien-ts with liver cirrhosis (see Table 5A).
I Where the solid phase conjugate combination was a pair of
5 ¦ 3-2B12 / rabbit anti-hPH Fab'-POD, the hP~I levels in sera from
healthy subjects and patients with liver cirrhosis were 95.3
and 151.5 ng/ml, respectively. Thus, significant difference
was observed between the two levels (p=0.001). Where the
combination was a pair of rabbit anti-hPH IgG / rabbit anti-hPH
10 ~ Eab'-POD the corresponding levels were 92.5 and 142.9 ng/ml,
~ respectively, thus showing significant difference (p=0.05).
; ¦ Example 4
I¦ One step assay of hPH using a polyvinyl chloride microtitration
¦¦ plate (Costar) as solid phase
15 ¦i 200 ~1 of 100 ~g/ml monoclonal antibody (3-2B12) was added
l to each well of a 96-weIl polyvinyl plate, and the plate was
i allowed to stand at 4~C to coat the wells with the antibody.
In carrying out assays, the wells were washed with diluting
buffer ~10 ~ phosphate buffer solution, pH 7.0, containing
0.1% ssA and 0.1 M sodium chloride). 20 ~1 each of a 0, 0.2, 0~4,
0.8,~1.6, 3.2, 6.4 and 12.8 ng/20 ~1 dilutions of the standard
hPH purified in Example l(a) and 20 ~1 of serum were indlvidually
added to the wells simultaneously with 150 ~1 of 100 ng/150 ~1
rabbit anti-hPH Eab'-POD conjugate in the diluting buffer
¦ 25 solution described abo~e. The wells were allowed to stand at
37C for 2 hours (first reaction). They were then washed three
times with 220 ~1 of lQ m~ phospha-te buffer solution, pH 7.0,
containing 0.1 M sodium chloride. 150 ~1 of 0.0134% TMBZ
dissolved in 0.1 M acetate huffer solution, p~ 5.5, and 50 ~1
1 30 of 0.01~ aqueous hydrogen peroxide solution were added/ and the~
.,
.
; ~'' ~ '~ '' .
- 27 ~5~'~4
plate was incubated at 37~C for 4S minutes (enzymatic reaction).
180 ~1 of each reaction solution was transferred into a test
tube containing 270 ~1 of 1.33 N sulfuric acid to stop the
reaction. A~ter termination of the reaction, the absorbance
at 450 nm was measured with a Shimadzu*double beam spectrophoto-
meter using water as a control. Using semi-logarithmic graph
paper with the abscissa indicating the amount in ng of the hPH
and the ordinate indicating the absorbance, all the data were
plotted to draw a standard curve. The amount oE hPH per ml of
a sample was calculated by multiplying by 100 the reading from
the standard curve of the hPH corresponding to the absorbance
obtained with 20 ~1 of the sample. In Fig. 4 in the accompanying
dxawings is shown an hPH standard curve obtained when a monoclonal
antibody (3-2B12) was used to coat a 96-well polyvinyl plate and
rabbit anti-hPH Fab'-POD was used as conjugate~ As is shown in
~'ig. 4, a curved line is observed in the range o 0-12.8 ng, and
the sensitivity was about 0.1 ng. The hPH level in serum was
measured for three healthy subjects using this standard curve
to give 49.32 and 18 ng/ml serum.
This one step assay technique is suit~ble for quantita~ e
or ~ualitative analysis where a great number of samples are
needed to be processed, although somewhat inferior in precision
to the sandwich two step assay technique using polystyrene balls
as a solid support.
Example 5
Identification o~ antigen
The following experimentation was conducted to ascertain
whether the antigen captured by the sandwich enzyme ir~unoassay
was identic~l with the hPH isolated in pure orm rom placenta
~n ~xample l(a~ . Thus, 1 ml o~ Sexum ~rom a p~t~ent with hepatic
Trade Mark
.: ` :.- ~ ` :
, .. .~.
, ' ~ ~ :
~ S~7'-~4
l disorder (hPH, 298 ng/ml) was subjected on an Ultrogel AcA 34
I gel filtration column (1.5 x 45 cm~ equilibrated with 0.l M
phosphate buffer solution, p~ 7Ø Fig. 5 in the accompanying
drawings shows an elution profile obtained by subjecting
human serum on an Ultrogel AcA 34 gel filtration column. 1n
Fig. 5, ( ) is for the absorbance at 280 nm and (-o-) is for
th~ hPH level measured with a pair of monoclonal antibody
(3-2Bl2) as a solid phase and rabbit anti-hPH Fab'-POD as a
conjugate. As shown in Fig. 5, two peaks of hPH level were
eluted at respective (Ve/Vo) values of approximately l.38 and
l~96. These fractions abundant in hPH were pooled and gel
filtration as described above was repeated twice thereto to
collect two fractions rich in hPH content (corresponding to 3 ml
of serum). The fractions were concentrated by ultrafiltration.
15 I The concentrated samples described above were subjected
¦ to affinity chromatography on a monoclonal antibody (3-2Bl2)-
¦ coupled Sepharose 4B column (0.35 x l0 cm, 52.4 ng antibody /
40 ~l gel) to purify hPH in the respective fractions. Thus,
the concentrated sample was adsorbed onto the above described
affinity column equilibrated with 0.l M phosphate buffer
solution, pH 7.0, and the column was washed with 6 ml of the
equilibrating buf~er solution. The adsorbate in column was
then eluted with 300 ~l of 0~l M glycine-HCl buffer solution,
pH 2.5 into a reservoir containing 300 ~l of 2 M Tris-HCl
buffer solution, p~ 8Ø The thus obtained effluents which
wexe deri~ed from the two fractions were dialyzed against
0.l M phosphate buffer solution, pH 7.0, concentrated and then
subjected to SDS-PA~ in accordance with the method of Baum
et al. described in Example l(a). After performing SDS~PAGE,
proteins was transferred from the acrylamide gel to nitro-
,
l~S~ ~ ~
cellulose shee-t (Trans-Blot M, 0.45 ~m, Bio-Rad) by the wes-tern
blotting method of Towbin et al. described in Example l(h).
This nitrocellulose sheet was treated with a monocLonal antibody
~ IgG (3-2B12)-POD, (specifically reactive with B-subunits, see
5 ~ Table 1), 3,3' diaminobenæidine and aqueous hydrogen peroxide
solution. Fig. 6 in the accompanying drawings shows patterns
obtained when two hPH-rich fractions shown in Fig~ 5 were each
~ purified on a monoclonal antibody (3-2B12) coupled Sepharose
¦ 4B affinity column, subjected to S~S-PAGE and then transferred
10 I to nitrocellulose sheet. In Fig. 6, (a) is for pu~rified
¦ hPH, (b) for F27-41 fractions in Fig. 5 and (c) for F45-59
fractions in Fig. 5. As reference molecular weights in this
experimentation was used a protein marker kit (BRL) containing
myosin H chain (200,000), phosphorylase b(92,500~, BSA (68,500),
ovalbumin (43,000),a chymotrypsinogen (25,700), ~-lactoglobulin
(18,400) and cytochrome C (12,300). As shown in Fig. 6, a main
band corresponding to ~-subunit with molecular weight of 60 KD,
and another band corresponding to molecular weight of 170-190 KD
were observed with the hPH derived from placenta (see Fig. 6-a).
On the other hand, with the 2 fractions corresponding to F27-41
and F45-59 in Fig. 5, there were observed, in addition to a band
corresponding to molecular weight of 60 KD, a few bands in the
higher molecular region (see Fig. 6-c).
hPH in tissues is said to exist in the form of a tetramer
(a2~2) wlth a molecular weight of 240,000 and consisting of a
(64 KD) and ~ (60 KD) subunits. Although, as clearly shown in
Fig. 5, hPH undergoes partial decomposition in blood and there-
fore its ex~ct subunit composition is not clear, the electro-
phoretic pattern in Fig. 6 sugyests that monoclonal antibody
(3-2B12) used as solid phase of the immunoassay captured intact
or partially decomposed hPH.
.
. - .,::
' ' . .
- ~
- :' ' '
~;S7 ~
¦ Table 1
_ . _ _ . .
Immunocross-
reaction with
Clone Isotype Chain subunit
2-lC2 IgG2b y2b/~
2-5G8 IgGl yl/K
2-6G9 IgA
2-7F8 IgA
3-2B12 IgGl yl/~
3-3H9 IgGl yl/K ~
3-4H2 IgM ~/~ ~ .
3-6H5 IgGl yl/~ ~ .
. ............ ... ~
:! 30 - .
.
A
,~ a) ~; r~ ~ I I , Z r~ c~
c~ r~ I rd u~ r/' r~ ~r~ !
I ~o ~ z ~ c r~ o ~ ¦ ~ ~ æ ~ 3 ~ t
_ ~ _---~
5 ~
æ ~ I ,~ I I I o I I I I ~ Z a~ I ` I ¦ ~ v
v ri Z ~ 3~ ~ ~ ~ r~ C ~ I ~i v ~
~ _ , ~ _ I osa
O ~`1 C ~ I O ~1 W ~1) ~1 I`i 1 _i 0 0 1 1 1 1 S ~ N
I s .~ :æ ,' cl I s r~ ~ 01 , ~D C~ C O
,. r_ v~ I C _ I _ r~ O ~
nI n ~; ~ 3l: a ,~ ~ o O ~ ~ ~.
û~ æ ~ c o o o l û~ __ z ~, ~ O ,~ ~ ~ca
a u~ ~ I co ~ j I q Z ~ ~ o ~ ~ o o I I
.~ u
~ 3 v 0 ~ o ~ u 1l c~ ~ ~ ~ ~ c~ Il ~ ~ a
u ~ z OO O OO U ~1 :~U O OO I II I ~aV
u~ -- o o ~o co o _I c ~ I r~ ~n _ l ~ C
~ct Z ~ co~ i ~ . ~c ~ Iol I
c ~t ~o 1~ o cr~ ~O I O _ ~ a~ ~ l c ~ ,
z: ~1 v~ ru~ æ ~ u~ ~ ~ ~ ~ u
1~ ~t ~o a~ r I o e' ~ U ~ r~ ~ o
~ __ . . ~ ~ _ _.___ I ~a ~ ~1
~ ~/ V~e col~o~ ! ~ ~ æ ,~,~c~i! I ,~0
E~ C) ~ u~ J ~ u~ ~ r~ o O o ~t ~ V ~t ~ u ~ r, ~ I v Oo
o l z Q~uc~ ~o I ~ o~oo~! I cO l ~ ua~ I I 1 I ~! ~ ~ ~,c
~ ~ ~I CO r~l I ~ -rl r-l ~ ~1 I
_ _ _. I__ _ I _
u ' ~ C~ ¢ ~.~ 0 U / ~, , , ~ ~ ~ ~1
~ / a c~ / ~ 7 _
/a ~ ~o~ U~~ ~ c co
C U
.~ / r _ I I I ~1O rl I t 1~ ~t 1.~ U / W _ U~ 0 r~ ~ l~o u
.
-- 31 ~
.
.. . . :
'
~L~65t7 ~'~
- - - --
z;
\ O ,~ ,-1 0
u~ c o o o o r
a) ~
u~ ~n ~ In
,~ h C o o o o
,~
~ ~ z _l C
O
z a~ D ~ ~ .
c ~ a) . . . . . ~,_
C o o o o o ~C h
~ ~co 1~ ~ O~r r~O a
--a~ ~ u,.,~ . . . . . ~a ~ ~ o
u~ ~ r~ ~ ~ o ~ o ,~ o ~ o ~ C
-- ~ 3: h C ~ ~O ,~
~D O a)
~ ~ I ~ ~a~ ~ ~ t~ h C
C r~ ~ Z ,~ ~. . . . . U~ C
''~ C ~'r~O ~ O ~ ~ ~ I~C rJ
n ".
C c) u~ h C u~ E~
,~ Z a~ D ~ ~ O
~a \ ~ . . . . . 3~1 ~
O u~ t) ~ o o o o ~ C ~
O ~ C u ~ t~ O ~ I
,~ I ~ ~ t~
u~ In
rl ~ U~ ~ ~rl h ~1
C~ H a~ O U~,~ o O ~ o Q h ,a U)
a) ~ h C ~ O ~ o ,~
Q. ~ t~ O a) u, ~ ~ h ~1
co oa~ coco 0 0
O ~ Z ~ C . . . . . ~ h C) ~ Q
'C~ Q ~4 ,1 o ~ o,~ ~S ::~ a) h
C-r~ _ ~ . ~ O U~-rl a
~:: C
~ Z a~ ~ ~ a) ~3 a
,_ ~ \ a) . . . . . O a) ~:: h
Z ~ C)o ~ ~ ~ O, S O ~ S ~
~1 a) ~ ~a h ~ U~
ti- ~ C) ~~ ~ r ~ sl O ~
C O ~ U~ ~ ~ O ~ S~ ~
,1 ~ ~ ~ U)o ~ o ~ O : o ~ O O
rd c~ m ~ ~
C O t~ O O ~I C ) h
rl Ç 1 : ~ ~L ) ~r~ c~ C~ o ,~
Q Q ~ ~ . . . . . ~ I~ O ~
k ~ rlO ~1 0 0 0 ~ ~ S O ~)
¦ h ~ (~
P~ z ~ a) ~ ~ ~ cO
: ; C~,C, ~a) ooooo ~Oou,
4~ rl :C O C ~ a)
.,~ c~ ~) ~I r~l ~ O r~ h ~1
a) (li t/~ U~ rl ~ ;:L ~ O ~1 0
O ~ ~ C~ ~ ~ o o o ,~ 1~ Q~ U Qr~`
: ~ c ~ o a) ~ c~ O
~ O l ~ ~~ I` ~ O o C ~ o C
E~ Z ~ Z ,~ C. . . . . ,~
: ~o o o l-
i .. ~
~ / ~
C~ ~ C~
~ ~ / ~
tTl / H H H H H
_l / I
rl /
C / '~
o ~ ,, a) co ~ ~ ~ c~
U/ ~ C c~ q ~C C~
/ O O u~
/ ~ ~ l l l l l
U ~ 1
, . ~_
..
- 32 -
. . .
::
. . ` ;~ `
, `: - : ."
` .: , . ,
` : ...
- `: ::- . ...
: `
...
I
o
L I ~ O Ln In O ~ J O I Ln
~f ~ I r~
o u~ ! ~,
l .,, ~P l ~ o.,~
_ U~ L~ ~ ~) r-l ~) ~ C~ r; r r~ v S
~4 _ a) ~ ~,) O r~l O O O O O 0 I t~l Q ~ O
O I LI rl ~ l ~ h
3:: ~ u~ Q a) :~ l C) O
5~ _1 0,~ l ,~
, 1 Q 1~ ~ a) l ~ U LLOI O
~4 ;LI _~ 0~ a) l Q o
~ ta ~? , ~ ~ ~;~ O ~
.q~ ~ ~ o o o ! o ~c`~Q
~ O ~ ~ Q ! ~ o, o ~
l ~oo,~
l 3 ~ L~ td
__ O ~ ~ O ,~ h
-~ z; ~D r; O Ll') CO O ~D CO I r a) ~ o
r~ U~ ~ Ln ~ ~ ~ Q h
~ ,~ I ~ ~ E
o ~ l s ~ o
~4 ~ l r~
t~ L C ~ r r~ L~ ~0
H _ V ~ a\0:~ o c;~ Ln CO ~ 1- ~ I ~ 1~ ) V (~
O tl Qa) ~ l E O
V~ ~ OS ~ 0
u~,l ~
l C~) tl) U~ l L~ a) U~
rl ~ V_ ~ l
H r~ ~; O C) l E~ O ~ l
Q) ~H _ _~ O . ::~ Q~rl ,a
~: ,~ ~ . ~ ~ v ~n Q
: ~ V ~ ~! 2~ r l ~ rl ~ n ~ ~ LL~ ,) 0 Ll
,Q~ C ~ O ~ ll r .C- ~
E~__ .___ .~ ~ LI-I h
~ / '~ ~:
,~ / )~ 1 ~1 ~I H 1~ H H I H
u I . .. _ !
~ rO ~ ~ U $ C~
I ~ u ~
_ .. .., .
~ .
.
., - ~ . . .... .~.
.. ..
~ '7~1~
J~ __
u ~ 1~
~: U ~ U ~_
,.~ ~ o 4~ o
.~ . .~ .
~o ~o
a u~ ~ ~
.,, ~
~n
3 h
~'u ~1 a~
. ~ ,~' ~
l ~ ~o ~
\
.
r~) o'
.C U ~ ~
ul~ R ~ t I
?:~ ~n __
. ~ ., ~ .
~
.a) ~ ' ~ a :
~ ~ R ~ a
~ ~1 ~ ~
'~q~
.. . .
.
,, ,
lZ~i5~ 4
t is understood that the preceding representative examples
may be varied within the scope of the present invention, both as
¦ -to the reagents and immunoassay conditions, by one skilled in the
~ ar-t to achieve essentially the same results.
¦ As many widely different embodiments of this invention
¦ may be made without departing from the spirit and scope thereof,
it is to be understood that this invention is not limited to
the specific embodiments thereof except as defined in the appended
claims.
I
, ~.
:
.. , , . . : :
:.:: . . , :
