Note: Descriptions are shown in the official language in which they were submitted.
1268~
TITLE OF TIIE INVENTION:
Substance-conjugated Complement Component Clq
BACI~GROUND OF T~IE INYENTION:
Field o~ the Invention;
This invention relates to a substance-conjugated
complement component Clq and a process for preparing the same.
~ore particularly, it relates -to a complement component Clq
conjugated with various substances including markers and cell
function regulating substances and a process ~or preparing the
same.
Related Art Statement;
It has hitherto been known to utilize the complement
fixation reaction for the measurement or determination of
antibodies in blood serum and anti~ens, such as microorganisms,
phisiologically active substances and chemicals~ This known
method makes use of the serial reactions wherein complement
components C1 to C9 are bound successively to an antibody
specifically bound to an antigen. In detail, -this kno~n method
comprises the step of adding an excess amount of complement
componen-ts to the formed antigen-antibody complex, the step of
determining an amount of residual complement components through
the hemolysis, and the step o-f determining the amount of
fixed complement components ~rom the degree of hemolysis.
The quantitY o~ the antigen or antibody is then estimated from
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126~3418
the resul-ts of -the amount of the fixed complement componen-ts.
In the hemolysis, complement components ac-t on the sensitized
erythrocytes including sheep red blood cells and an-ti-sheep red
blood cell antisera so that -the complement components may
be de-termined while using the hemolysis o-f the sheep red blood
cells as the index. However, practical de-termination operation
of the hemolysis is extremely complex and needs high level skill
and knowledge. In addi-tion, this known method has a relatively
low sensitivity and requires two days for the determination
- lO operation.
Various methods have been proposed -to overcome the
aforementioned disadvantages of the known method as described
in the preceding paragraph. For example, Japanese Patent
Laid-Open Publication No. 43498~1980 discloses one of such
` 15 methods. In the method proposed by the antecedent Publication
referred to above, an antibody which binds, as an antigen,
a complement component being bound to another antibody is
labelled wi-th an enzyme, and the amount of the thus labelled
antibody is determined by the enzymatic activitY thereof.
This method is, therefore, one of the so-called enzyme-labelled
antibody techniques. However, this me~hod involves two step
reactions~ since a labelled antibody which binds, as an antigen,
a complement compQnent must be used. Accordingly, rinsing
operations are required after each of the reactions, leading
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12684~8
to increase in labor and time. In fact, this determination
method costs much time as several hours.
On the o-ther hand, a method of determining a
neutralizing antibody has been made known, for example,
by Takashi Kitamura, "Tissue Culture Technology for
Inspection of Virus", published by KINDAI SHUPPAN (1980~,
page 246. When an antibody against poliovirus, -for instance,
is determined by this method, cultivated cells originated from
human being, a monkey or an ape are -first inoculated with the
poliovirus. (Meanwhile, the poliovirus does never gro~ if it
is inoculated into cells originated from the sources other than
human being, a monkey or an ape.) The cells inoculated with
the poliovirus collapse and are deseased as the result of
cytopathogenesis due to propagation of the virus. However, the
reaction product of a neutralizing antibody and the virus,
(the infectiousness of virus being neutralized by the
neutralizing antibodY), can no-t propagate even if it is
inoculated upon a cell originated from human being or monkey so
that the cell is kept to have normal form and functions. Making
2Q use of this principle, a specific virus is reacted with blood
serum and then the titre of the neutralizing antibodY is
determined by inspecting the presence or absence, and -the degree
i-f present,of plaque and CPE (cytopathogenic effect~.
Ho~ever, when the poliovirus~is determined by the
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1~684~8
method described in the preceding para~raph, the poliovirus
mus-t be cul-tivated for abou-t 7 days in a normal tes-t in
addition to -the fact that -the inspec-tion and judgemen-t of the
result should be made by a skilled person rather than being
- 5 easily conducted by a person having ordinary or middle level
skill. For this reason, an order of test for the determination
of neutrali2ing antibody is not accepted even by a large scale
inspec-tion cen-ter at the present day.
On the other hand, as a method for determining
antigens or an-tibodies in a simpler way, there has been known
- in the art a method wherein properties of complemen-t component
Clq binding an antigen-antibody complex is u-tilized.
(Simpson e-t al.,"Jounal of Immunological Methods", Vol. 67,
167 to 172 (1984~.) In this kno~n method, glutalaldehyde or
periodic acid is conjugated to the complement component Clq
as a cross-linker, and peroxidase (en~yme~ is coniugated via
said cross-linker to the complement component Clq as a marker.
Marchalonis J. J., "Biochemical Journal", Vol. 113, pp229 to 305
(1969) discloses a method in which radioactive iodine is
conjugated to the complement component Clq through the
chloramine T me-thod as a marker. However, in these known
; methods, an enzyme or radioactive iodine is coupled with each
of the complement component Clq molecules via an amino group
present on the molecule generally and at random, resulting in
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~2684~8
entire nlodi-fication of the molecule since the very site o~
each molecule having inheren-t properties capable of binding to
an immunocomplex has been chemically modi~ied by said cross-
linker or coupler. Accordingly, the binding activity of such a
marker-labelled complement component Clq for binding to an
antigen-antibody complex is seriously lowered to a level not
to adapt for quantitative measuremellt as a reagent. Moreover,
a false-positive reaction takes place frequently by the latent
presence of said cross-linker in the marker-labelled complement
component Clq to make i-t impossible to continue the determina-
~; tion operations. It has, thus, been impossible to provide a
reliable determination method for determining an antigen or
~: antibody in a precise and reproducible manner by the use of
the complement component Clq.
OBJECTS AND SUMMhRY OF THE INVENTION:
It is, therefore~ a primary object of this invention
to provide a complement component Clq which is conjugated
with a variety of substances, such as markers or cell function
regulating substances, while preserving its inherent bindin~
capacity for binding with an immunocomplex without any
detraction, and a process for preparing such a complement
component Clq.
Another obJect of this invention is to prov;de a
complement component Clq which is conjugated with a vriety
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126~34~8
of substances -to be conveniently used as a determination or
detection reagent for determining or detecting a speci~ic
an-tigen prese~-t in a body fluid or held or bound to a cell or
body tissue or for de-termining or detecting the corresponding
antibody for the specific antigen, a modified immunoglobulin or
immunocomplex, and a process for prleparing such a complement
component Glq.
A fur-ther object of this invention is to provide a
complement component Clq which is conjugated with a variety
of substances to be convenientlY used as a curing agent or
. medicine for regulating physiological function of a variety
of cells which have cell surface structures identi~ied by
specific antibodies or which can capture specific
immunocomplexes or complements, and a process for preparing
such a complement component Clq.
The above and other objects of this inven~ion will
be apparent from the following detailed description thereof,
According to the present invention~ there is provided
a complemsnt component Clq wherein a substance is conjugated
via a sulfur atom to at least one site of said component, said
site being not involved in binding immunoglobulins.
Also provided in accordance with this invention is
a process for preparing a substance-conjugated complement
~` component Clq, comprising the steps of:
:
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~8418
(a~ adding a reducing agent to a complemen-t component Clq
to sleave at least one S-S bond presen-t at a site not involved
in binding immunoglobulins thereby to obtain a reduced
complement CoDIponent Clq having at least one exposed -SH
group; and
(b) conjugating a substance to said complement component
Clq via said exposed -SH group.
BRIEF DESCRIPTION OF THE DRAWING:
The sigle figure, Fig. 1, of the appended drawing
is a graph showing the change in neutralizing antibody titer of
an an-ti-HSV positive human serum in Example 15, one of the
examples of the invention.
DESCRIPTION OF THE INVENTION:
The present invention will no~ be decribed in detail.
In general, after an antigen is bound to an antibodY,
a complement is bound to the antibody already bound ~ith the
antigen to destruct the antigen. The complement includes
complement components Clq, Cls, Clr, C~a, C2b, C3a, C3b, C4b?
C5b, C6, C7, C8 and C9, and each one of these complement
; 20 components binds to a specifmc antibodY pertinently dePending on
the specific immunological reaction or allergy rection. These
complement components bind in a fixed order such that the
component Clq binds to the antigen at a first place, follo~ed
by binding of Cls and Clr to Clq, and -then -the other complement
- 7 -
12~i8418
components bind serially.
After eager investigations with the estimation that
a substance-coniugated complement component having a utility
when used as a determination reagent or a curing agent might
;~ 5 be prepared by conjugating a variety of substances to the
t~
complement component Clq, which is the component coupled with
an antigen-antibody complex at the first place, such that the
subsequent binding between the immunoglobulins acting as an
antibody and the component Clq is not hindered, we have
succeeded to allow a variety of substances to conjugate at
sites of the complement component Clq other than the sites at
which the immunoglobulins are to be bound.
In detail, we have given attention to the presence of
; nine S-S bonds located at the sites of -the polypeptide molecule
of the complement component Clq, the S-S bonds locating at the
sites remote enough for affecting -the site having the binding
capability for the immunoglobulins and being formed by the
fourth cysteine residues from the N-terminals of the h, B and
C chains of the polypeptide constituting the complement
component Clq so that they are apt to be attaked by a reducing
agent and apt to coniuga-te with a substance readily.
In view of the presence of such S-S bonds, we have
contemplated to cleave these S-S bonds by the action of a
reducing agent to expose at leat one S-}l group in a step ~a) of
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126~34~8
the process provided by the presen-t inven-tion.
The reducing agen-ts which may be conveniently used
in this step ~a) include -tl~ose used conven-tionally, the
examples being sulfur-containing compounds, such as
mercaptoe-thylamine, di-thiothreitol, 2-mercaptoethanol,
cys-teine and glutathione.
The reducing step is carried ou-t under -the condi-tion
that the complement component Clq is not modified. Preferably,
~` reducing may be effected by dissolving the complement component
Clq in a buffer solution in which it exists stably and then it
is a-ttacked by a reducing agent. Examples of the buffer
solution used for this purpose include a tris buffered saline
containing 10~ of sucrose, 1 mol of sodium chloride and 5 mM
(millimols) of sodium ethylenediamine tetra-acetate, and a
; 15 phosphate buffered saline (PBS). The reducing reaction is
carried out, generally, at about -2C to 45C for about 30
seconds to 24 hours, -the reaction temperature and time being
changed depending on the specific reducing agent used.
It is desirous that the thus reduced complement
component Clq be s$ored in a buffer solution to be used in the
subsequent step (b) after removing the excess reducing agent
by means of a conventional method, such as dialysis, salting-out
process or gel filtration.
The complement componen-t Clq utilized in the present
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126~34~8
invention is a glycoprotein contained in the blood serum of
animal, and has nature for binding firmly to the immunoglobulins
when the immunoglobulins contained similarly in the blood serum
and acting as an antibody react specifically with the
corresponding antigen. The complement component Clq used in the
invention may be isolated from various aminals including sheep,
rabbit, guinea pig, cattle, horse, dog, mouse and human being,
and the frac-tion enriched in the Clq component may be picked up
in accordance with a conventional purifying operation. (In this
connection, reference should be made to "Operations in
Immunological Experiment B", published by the Japanese
Immunological Society, pp 137~ to 1380 (1974), if necessary )
In the process of the invention, a variety of
substances is conjugated via the exposed SH group of the
reduced complement component Clq at the subsequent step (b~.
The substances to be used in the step (b) and to be conjugated
` with the complement component Clq via the exposed SH group
-` include signal emitting substances, such as en~yme substrates,
dyestuffs, ma~netizable substances, donors or acceptors for
electron transference, radioactive materials, :etal compounds
and metal compositions, which emit signals detectable by -the
sensory organs or external ins-truments, or en~ymes or coenzymes
~hich may be modified to emit detectable signals; cell
function regulating substances, for example, certain en~ymes
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which act on the counter-substances conjugated to the complement
componen-t Clq -to provide the latter with any functions; and
substances, such as high PolYmer ma-terials, which capture
or fix the counter-substances coniugated to the complement
component Clq.
More specifically, examples of the enzyme substrate
are o-nitrophsnyl- ~-D-galactopyranoside and 3-hydroxYsteroid;
and dye stuffs include the redox dyestuffs, such as methylene
Blue, and fluorescent dyestuffs, such as fluorescein isothio-
cyanate. Examples of the magnetizable substance are organic
irons, such as carbonic iron, and microcapsules containing
iron, and complexes of iron with proteins may also be used.
The donors and acceptors for electron transference include
a ~ide variety of substances which take part in the electron
transference, and chlorophyll which may be energized to take
part in the electron transference is included in this group
o-f substance and preferably used in the invention. Examples
of the radioactive substances are 1Z4I-labelled albumin,
`; p-chloro(203Hg)mercuriben~oic acid, N-ethyl~2,3-14C~maleimide
and iode(1-14C)acetamide. ;
The metal compounds and compositions, other than the
corbonic iron referred to hereinabove~ ~hich may be used in the
~ invention include gold colloid and iron-containing microbeads~
`~ Examples of the en~ymes are peroxmdase, alkaline
lX~8418
Rhospha-tase, galac-tosidase and alcohol dehydrogenase; wheras
examples o~ the coenzymes are nicotinamide adenine dinucleotide,
nicotinamide adenine dinucleotide phosphate, flavin adenine
dinucleotide and flavin adenine dinucleo-tide phosphate.
A varie-ty of substances may be included in the cell
function regula-ting substance and conveniently used in the
inven-tion, examples being surface active agents; antibiotics
having activities to vi-tal membranes, such as ampho-tericin
B, and antibiotics affecting the metabolisms of cells, such as
actinomycin D; trace essential nutrients or growth factors
indispensable ~or the growth of cells, such as selenium
compounds, insulin, -transferrin and epidermal ~row-th fac-tor;
hormones such as corticosteroid; factors participating in the
manifestation of cell function, such as macrophage activation
factor, factors participating in the cell division, such as B
cell division factor; anticancer agents, scuh as mitomycin C;
and toxins such as ricin of toxin o~ castor bean.
For instance, an enzyme may be utilized as the signal
emitting substance while being conjugated to the complement
component Clq to act as a marker so that it is used as a
determina-tion or detection reagent for determining or
detec-ting a specific antigen present in a body fluid or held
by or adhering to a cell or body tissue, or for de-termining or
detec-ting the corresponding antibody -to said antigen, modified
- 12 -
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immunoglobulin or an immunocomPlex.
When an antibio-tic is used as the cell function
regulating substance while being conjugated to -the complement
componen-t Clq, the conjugated product may be used as a curing
agent for controlling the physiological function of a cell
having a cell surface structure which can be identified
by a specific antibody or a cell capable of capturing an
immunocomplex or a complement.
In the a-forementioned step ~b) of the process of the
invention, a substance having a group which can be conjugated
to the exposed -SH group may be directly conjugated to the
reduced complement component Clq, or a substance may be
conjugated indirectly to the complement component Clq via a
second substance having a coupling function, for example, via
one or more spacers or cross-linkers. E~amples of the substance
which may be directly conjugated include activated thiol
7~Rqd~rl~
Sepharose ~rah~-~a~r of Pharmacia Fine Chemicals Co.) and
p-chloromercuriben~oate. Any substance having a group
capable of conjugating to the e~posed -SH group of the
complemen-t component Clq, such as maleimide~residue or -SH
group, and also having another group capable of coupling with
the substance to be coupled indirectly with the complement
component Clq maY be used as the coupling agent for such purpose.
The coupling agent or the second substance haYing
- 13
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a coupling func-tion, of course, varies depending on the
substance to be coupled thereby. As an illustra-tive example,
~hen peroxidase ex-tracted ~rom horseradich is coupled by
the N-hydroxysucc:ineimide ester of N-(4-carboxycyclohexyl-
methyl)maleimide, both reagents are dissolved in a bufferhaving a pH value of 6.5 to 7.5 and containing sodium
ethylenediamine tetra-acetate to allow -them to react wi-th eath
other at 30C for an hour. The reac-tion conditions are selected
in consideration of the characteristics of a cross-linker used.
Other than the N-hydroxysuccineimide es-ter o~ N-(4-carboxycyclo-
hexylmethyl)maleimide re~erred to above, N-hydroxysuccineimide
esters of m-maleimide ben~oic acid, N-~4-carboxyphenylmethyl)-
maleimide and maleimide acetic acid may be used as the cross-
linker. A peroxidase having -therein a maleimide group is
lS produced The thus produced peroxidase having a maleimide
group is mixed with the reduced complement component Clq,
and maintained in a buffer held at pH 5.5 to 6.5 and containin~
sodium ethylenediamine tetra-acetate at 4C for 20 hours,
whereby a complement component Clq conjugated with peroxidase
is ob-tained.
At the final step, the fraction of the complemen-t
component Clq labelled with peroxidase, which serves as a
marker, and having the activities originated both from the
peroxidase and the complement component Clq may be picked up
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-through -the gel filtration.
It is preferred that the s-tep (b) o~ the process of
the invention be carried out in the presence of a buf~er for
both of the reduced complemen-t component Clq and the substance
to be conjugated thereto , and carried out in the presence of
a buffer for the coupler or cross-linker in case where such a
substance having the coupling or cr~ss-linking function is
used.
In the substance-coniugated complement component Clq
provided according to this invention, the substance is
; conjuga-ted at a site or sites having no bindin~ capacity with
` the immunoglobulins, so that the binding capabilty to the
immunoglobulins inheren-t to the complement component Clq is
kept intact without being hindered by the conJugating substance
Since the substance-conjugated complement components
Clq provided according to this invention are conjugated with
various signal emitting substances and cell ~unction regulating
` substances without bloching the sites at which the
; immunoglobulins is to be bound, they may be used for various
.
applications including determination or measurement rea~ents
giving the reproducible results or curing medicines in which
their inherent capacities for binding with -the immunoglobulins
are utilized.
The method ~or the measurement or determination,
- 15 -
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utilizing the substance-conjugated complemen-t component Clq will
now be described. The signal emit-ting substances, as described
hereinbefore, may be used in the measurement method according to
the inven-tion. Such a signal emitting substance may be u-tili7ed
as a marker. In detail, a substance-conjugated complement
component Clq having a marker conjugated at a site tha-t is no-t
adapted to bind with the immunoglobulins is prepared, and then
the thus prepared marker-labelled complement component Clq is
allowed to react with another material to be measured, whereby a
reaction product coniugated with the marker is obtained.
Subsequently, the labelled marker is qualitatively or
quantitatively analysed to measure a variety of antigens,
antibodies, neutralizing antibodies, substances produced or
appearing in cells or on the surfaces of cells or microorganisms.
In this manner, the method for measurement may be applied for
comprehensive uses, including various clinical inspections and
; diagnoses of diseases.
One group or category of the substances which
may be measured includes complement-binding antibodies. By the
determination of certain complement-binding antibodies, various
diseases of wide-ranging hosts including not only human beings
but also animals and plan-ts may be diagnosed and judgement may
; be made whether the hosts are infected or not, the diseases
which may be determined including infectious diseases
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caused by mic~oorganisms~ such as bacterium, chlamydia and
virus, tumor and autoimmune diseases such as systemic lupus
erythematodus. Par~icularly in clinical inspection of such
an infectious disease and autoimmune disease, it is a common
practice -to inspect whether or not ~ specific antibody uniquely
appearing with a certain disease is present in the blood serum.
Various measurement or inspection methods are kno~n, and -the
complement-binding reaction is involed in one of the impor-tant
inspection methods. Notwithstandin~ that this complement-
binding reac-tion has a utilitY when utilized in a method of
inspecting a certain specific antibody, the reaction has not
been frequently uesd, irrespective of the importance thereof,
since it involves cumbersome determination operations and
requires extremely high level skill.
Under these circumstances, a considerable contribution
in indus-trial and medical fields is made by the provision of
a simple and speedy method based on this principle is developed
to give a reproducible measurement or determination value at
high sensitivity.
In determination of a complement-binding antibody J an
antigen is initially fi~ed -to a solid phase or carrier, and then
the fixed antigen is allowed -to react with an antibody and ~ith
a complemen-t component Clq combined with a marker~ followed by
removal of unreacted materials and then -the 0arker is
- 17 -
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quan-titatively analysed. Utilizable antigens include viruses
and bacteria, such as varicella-zoster virus, measles virus,
rubella virus, in~luen~a virus, herpes simple~ virus, hepatitis
virus, mumps virus and mycoplasma phneumonitis; physiologically
active substances such as interferon; and antigens against
autoantibodies such as DNA. Initially, such an antigen is fixed
to a solid carrier to form a solid phase. Whereupon, the
operations, par-ticularly rinsing operations, can be carried out
easily as compared with the prior art technology in which a
lquid phase must be handled. Any carriers may be used as far as
the antigen absorbed thereby is not easily released from the
solid phase, the examples being synthetic high polymers such as
polyvinylchloride and polystyrene, natural high polymers such as
filter paper, and cells and tissues. More specifically, a
- 15 microtiter plate and polystyrene beads may be referred to as
illustrative examples. The antigen may be fixed to the solid
phase by fi~ing the same on the sur~ace of the solid carrier
through physical absorption or chemical covalent bond and the
like. In case where a cell or tissue is used as a solid carrier,
the antigen may also be ~ixed thereto by infection.
Then, the fixed antigen is allowed to react with
the specific or corresponding antibody and a complement
component Clq combined with a marker. The antibody is the
objective substance which is to be measured or inspected, and
: `. :
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the examples thereof are body fluids, such as blood serum,
cerebrospinal fluid and saliva. The blood serum is used most
frequently, since it contains the largest quantities of
antibodies. The an-tibodies determined or measured must
bind to the corresponding antigens uniquely or specifically,
and must be capable o~ binding to the complement components.
However, almost all of the antibodies produced in living bodies
satisfy the aforementioned conditions, and hence they may be
measured.
The reaction of the antigen fixed to the solid phase,
the an-tibody and the complement component Clq conjugated ~ith
a marker takes place spontaneously by simply mixing the
aforementioned -three reactants together to complete the
reaction for forming a combined product. While the reaction
temperature and reaction time vary depending on the specific
antigen or other reactants used, these conditions may be
selected properly unless bioactivities are lost,
Since the complex composed of the antigen, the
antibody and the marker-labelled complement component Clq is
fixed to a solid phase, the unabsorbed complement component Clq
and inhibitors for the reaction can be readily removed by
rinsing. The marker incorporated in the comple~ is then
quantitatively analysed. For quan-titative analYsis, any of
the kno~n methods may be used, including visual measurement,
- 19 -
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observa-tions -through various types o~ microscopes, IDeasurement
of absorbance of visual and ultraviolet rays, fluoropho-tometric
measuremen-t and p~lse count measurement. In determination of
the marker7 other than -the direct measurement of the marker
labelled -to the combined complex~ a known quantity of the
marker is used and the quantity of the marker which has not
been conjugated to the complement component Clq may be
determined to learn indirectly the quantity thereof introduced
into the complex. Anyway, by the quanti-tative analysis of
the marker, the quantity of the complement-binding antibody
coupled to a specific antigen can be learned.
In the measurement or determination, enzymes are
` particularly preferred as the signal emitting substance. Since
- enzymes act as catalysts, the sensitivities of the measurement
may be freely adiusted by changing the temperature and time of
the reaction.
Various antigens may also been inspected. The
measurement of a variety of antigens may be utili~ed for the
detection, identification, quantitative analysis and inspection
of various substances including microorganisms such as chlamydia
and virus, various physiologically active substances such as
interferon and lymphokines, specific antigens for cancers~
specific antigen substances in immunological abnormalities,
allergens in allergic diseases, and medicinal substances such
.
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34~
as hor~ones; and thus the method may be applied for various
purposes, such as speedY and reliable diagnosis on a variety of
diseases, provision of the s-tandards for judging the effects
of curing actions, inspection for doping, inspection ~or the
determination of foreign matters in products, and hygienic or
sanitary inspections.
When a specific antigen is measured, a substance
having affinity with the antigen which substance is fixed to a
solid carrier is reacted with the antigen, and the complement
component Clq labelled ~ith a marker,and optinally with an
antidoby, followed by removal of unreac-ted materials and then
the marker is quantita-tively analyesd.
The substances having affinity with the antigen
include various types of substances by ~hich the antigens
are readily absorbed, examples thereof being antibodies,
; portions of antibodies containing the sites binding to the
antigens [for example, Fab, F~ab' ) or F(ab' )2]~ en~yme
substrates and inhibitors, protein A of staphylococcus, various
medicinal substances originated from organisms, and receptors
for virus. A living tissue containing a substance having
affini$y ~ith a specific antigen may be used without being
purified. A selected one of these substances ~aving af~inities
with antigens is fixed to the solid phase or carrier~ ~y the
use of the fixed phase, rinsing and other operations can
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be simplified. Any carriers may be used for this purpose,
as far as the substances having affinities with antigens are
not readily released or removed, and the same carrier materials
as has been described for the method of measuring -the
complement-binding antibodies may be used.
A specific antigen to be measured is then added to
the fixed substance having affinity with the antigen so that
the an-tigen is conjugated with the substance. ~ny antigens maY
be measured without particular limitation, as fas as they can be
coupled with substances having affinities thereto, such as the
corresponding antibodies. Illustative antigens which may be
measured include microorganisms such as viruses and bacteria,
products produced by viruses and bacteria, vital components in
animal tissues, physiologically active substances of plantst and
chemicals. Sources for such antigens are body fluids such as
blood, urine, cerebrospinal fluid and saliva, processed products
of meats and plants, and aqueous solutions from rivers, sewege
or waste water.
In the method of measuring an anti~en, the
corresponding antibody is added optionally as necessity arises.
When an antibody or a portion of antibody having an antibody-
binding activity and a complement-binding activity is used as
the substance having the affinity with the antigen, it is not
requisite to further add the antibody. However, in case where a
- 22 -
: :
,~.. : , :....... ~.
: :.. , :.
~26~
susbstance having no complement-binding activity is used as -the
substance having the af~inity with the antigen, it is essential
to add the an-tibody to be bound to the marker-labelled
complement componen-t Clq. The antibody may be added at any
desired time point after the antigen is added, and may be added
simultaneously with the addition of the complement component Clq
or may be added be~ore or after the addition of the complemen-t
component Clq. Of course, the an-tibody should be the one which
binds uniquely to the specific antigen to be measured and should
be capable o~ binding with the complement component Clq.
Typical antibodies used commonly are immunoglobulins con-tained
in animal blood sera, the examples being IgM, IgG, etc. having
`complement-binding activities. Natural antibodies present in
blood sera may be used, or desired antibodies may be obtained
by administrating or infecting animals with an-tigens. In
addition to the immunoglobulins purified and separated from
blood sera, inactivated blood sera may be used as the antibodies
without purification.
The condition ~or the reaction between -the antigen,
the substance having affini-ty with the antigen, and the marker-
labelled complement component Clq and the antibody if it is
added is not ristrictedO Only by mixing the ma-terials,
the reaction proceeds spontaneously and quantitatively. While
the time and temperature o~ the reaction vary depending on the
:
- 23 -
,,". ., - -:
~ .: . ~ ,. ; .
specific kinds of the antigen and the other reactants,
the reaction condition maY be set with the only limitation
that -the biological ac-tivities o~ the reactants are preserved.
Since the comlex of the substance having affinity with
the antigen, the antigen and the marker-labelled complement
component Clq is fixed to the solid phase, the unreacted
complemen-t component Clq and inhibitors for the reaction may be
easily removed by simple rinsing operation. The marker of the
complex fixed -to the solid phase is then quantitatively analysed.
For the quantitative analysis, similar methods as has been
described in determination of complement-binding antibody may be
employed.
Neutralizing antibodies may also be measured.
Neutralizin~ antibodies are antibodies for preventing infections
by microorganisms, such as virus, ricksttsia and chlamydia
Referring to diseases caused by virus, for instance, a wide
variety of viral infectious diseases have been known up to date,
including not a few serious diseases. For instance, i~ a
pregnant woman is infected with rubella virus, there arises a
danger that a malformed baby is born. Fatal damages are caused
by the infection with rabies virus, Japanese encephalitis virus
and poliovirus, with the nerval cells su~fered unrecoverable
disorders, leading to lasting troubles throughout the lifetime.
Hepatitis caused by hepati-tis virus is an infectious disease
.
~ - 24 -
.: ' ' - ,.. ' ~ !
: :: ' ' , '`. , ~' :
", : -';
''
~;8
which lasts as a chronic disease for a very long time, and a
portion of the liver is impaired by liver cirrhosis which might
lead to hepatoma.
Ho~ever, it is extremely hard to inhibit the growth
of virus by the use of a variety of medicines including
antibiotics, since a virus can grow in special living cells, i.e.
the susceptible cells, unlike bacterium and fungi~
Accordingly, it is a more important coun-ter-measure
against the diseases caused by virus to prevent infection by
virus or to protect a person from infection, apart from the
curing treatement of the patients. The judgement on the
question whether a person is susceptible to infection by a
specific virus or not may be rendered by -the determination of
presence or absence of the neutralizing antibody to the
virus under question and by the measurement of the titer of
the existing neutrali~ing antibodY.
~ living body acquires sound immunitY after it
has been infected with a specific virus and then recovered
from the disease caused thereby. This means, in fact, -that
a system for preventing the living body from re-infection
with that virus has been established. In other ~ords, a living
body has been once infected with a specific virus, -the antibodY
for pro-tecting the body from re-in-fection is promoted, the
antibody being referred to as infection preventing antibody.
- 25 -
,,.. , , . . , -
.- . . ..
- . :, -, ~
:: - ~ , . .
~2~841~3
Produc-tion and preservation of the infection preventing
an-tibody are very impor-tant fac-tors against the infection by
-the virus. After being infected or immunized with a virus
against the attack by the virus (for example inoculated by
vaccine), various antibodies a~ainst the s-truc-tural components
of the virus have been produced in a living body. However,
all of these antibodies produced in the living body are not
participated in the prevention against infection by that virus.
Only the antibody having the function for inhibiting the growth
or propagation of the virus is referred to as neutralizing
antibody or infection preventing antibody. This particular
neutralizing antibody exerts the principal role in prevention
of infection. There is provided a method of measuring a variety
of neutralizing antibodies a~ainst all viruses, rickettsias and
chlamydias which infect culture cells speedily and
quantitatively on a number of samples.
In the method of measuring a neutralizing antibody,
a liquid containing a known quantity of microorganism, such as
virus, rickettsia or chlamydia, is reacted ~ith a body fluid to
20 - be measured, such as blood, cerebrospinal fluid, saliva or blood
serum. ht this reaction step, the neutralizing antiboy, if
present, reacts ~ith the mixed microorganism . The amount of
microorganism reacting with the neutralizing an-tibody is
increased as the amount of neutralizing antibody contained-in
- 26 -
. . .
.
. .
;- . . ,
.
... ~: .
~;26~3418
the liquid under measurement increases so that the amount of -the
residual microorganism is decreased. Since the content of
microorganism in the measured liquid is known, -the amoun-t of
neu-tralizing an-tibody contained in the measured liquid can be
calcula-ted from the result of determination of the residual
microorganism.
After the preceding reaction step, the residual
microorganism is inoculated on culture host cells to allow to
grow. The cultivation is stopped after the lapse of pre-set
time, whereby fixed cells containing therein the residual
microorganism are obtained. A marker-labelled complement
componsnt Clq and an antibody against the microorganism are
then added to react with the fixed cell to obtain a modified
fixed cell to which the marker-labelled complement component
Clq and the antibody are bound. By the quantitative analysis
of the marker, the amount of residual microor~anism can be
learned to find the amount or tlter of the neutralizing antibody.
The residual microorganism is, in general, cultivated
initially by inoculating -the microorganism on hos-t cells
cultivated through a monolaYer culture on a micro plate to
allow the microorganism to be a~sorbed by the micro plate, and
then allowing it to grow or propagate on the plate. It is a
common practice to inactivate endogenous enzymes and -the
microorganism by treating with, for example, methanol-
- 27 -
. .
- . , . ;., . , . :
- :.~ , . . . .
- - . . -
. ,. ~ . ... ,~, . .
. , .. , . ~- :
~:6~3418
containing hydrogen peroxide. Antibodies against the residual
microorganism ~hich may be used in -the method include antisera,
such as lo~ titer human sera and animal immunoe sera, and
monoclonal antibody.
Fur-thermore, the materials ~hich may be measured,
other than those described above, are products produced or
appearing internally of or on the surfaces of cells,and various
microorganisms. The materials belonging to this category
include cell surface antigens produced by cells~ such as asialo
GMl, T antigen and Ly antigen; intracellular enzymes, such aæ
TdT (terminal deoxynucleotidyl transferase), GTP (~ -glutamyl
transpeptidase) and LDH (lactate dehydrogenase); secreting
substances, such as CEA (carcino embryonic antigen) and AFP (~ -
fetoprotein) and immunoglobulins; and enzymes and peptide base
substances produced by yeasts and bacteria. The cells referred
to above include all kinds of cells including animal cells,
plant cells, heterokaryotes, cells of yeasts, bacteria and
proto~oa, and cells subjected to ~ene engineering.
Microorganisms ~hich may be measured include all microorganisms
infecting cultivated cells, such as viruses, rickettsias and
chlamydias.
In measurement, one of the aforementioned substances
or microorganisms is cultivated and fixed, or simply fixed,
and then reac-ted with a complement-binding antibody and a
- 28 -
: ; -: .
- . " .
.. .
.,
. ' ' '. "'' '
. ,'. .,
~684~8
marker-labelled complemen-t component Clq, followed by
determination of the marker, whereby the substance or
microorganism may be quantitatively analysed.
EXAMPLES OF THE INVENTION:
The invention will now be illustratively described
with reference to examp]es thereof and comparative examples.
Example
Enzyme-Conjugated Complement Component Clq:
(1) Purification;
100 mQ of a fresh rabbit blood serum was dialized
through 5 Q of a 0.026M aqueous solution of ethylene glycol
tetra-acetate ~pH 7.5) for 15 to 24 hours, and the formed
precipitate was recovered by centrifugal separation (20,000G,
20 minutes). The recovered precipitate was dissolved in 20 mQ
of a 0.75M aqueous solution of sodium chloride (pH 5.0)
containing O.lM of sodium ethylenediamine tetra-acetate.
After removing the insoluble materials by centrifugal
separation (25,000G, 30 minutes), and then the solution was
dialized through 5Q of 0.063M aqueous solution of sodium
ethylenediamine tetra-acetate (pH 5.0) at 5C for 4 hours,
followed by removal of precipitates by centrifugal separation
(20,000G, 20 minutes)~ About 3 mg of pro-teins were obtained
by the aforementioned operations, and 95~ or more of the thus
obtained proteins was occupied by the complement component Clq.
- 29 -
.
.. - :. , .
" : : ..
- -. ~ . ,.
: ~ . , . .... :
~268~
In order to store the complement component Clq~ the proteins
were dissolved in an aqueous solu-tion (pH 7.4) containing
O.O~M tris(hydroxymethyl)aminomethane, lM sodium chloride,
0.005M sodium ethylenediamine tetra-aceta-te and 10X sucrose.
The aforementioned operation sequence maY be repea-ted to
further purify the complement component Clq.
(2) Preparation;
30 m~ of the thus purified rabbit complement component
Clq was dissolved in 10 m Q of an aqueous solution (pH 7.4)
containing 0.05M tris(hydroxymethyl)aminomethane, lM sodium
chloride, 0.005M sodium ethylenediamine tetra-acetate and lOX
sucrose. The solution was then added ~ith 0.1 mQ of a O.lM
dithiothreitol, and allowed to stand at room temperature for
an hour for reaction. The reaction solution was then passed
~ r4 ~ e ~
through a Sephadex G-25 ~rr~e-y~-e of Pharmacia Fine Chemicals
Co.) column to recover the protein fraction which was
concentrated to have a volume of about 10 mQ by ultrafiltration
to obtain 22 mg of a reduced complement component Clq.
Separately, 20 m~ of peroxidase extracted from the
horseradish was dissolved in ~ m Q of a phosphate buffer
(pH 7.4)~ and then added with 4 mQ of dimethylformamide.
The solution was further added with 0.2 mQ of a 2X
4-(maleimidemethyl)-cyclohexane-1-carboxylic acid succineimide
ester (hereinafter referred to as CHM) in dimethylformamide,
- 30 -
.
: "; , :
~ ~ : -, -, '' - ,: `' .. .
,
~2684~8
and then allowed to stand at room -temperature for an hour for
reaction. After an hour, the solution con-taining the reaction
product was passed through a Sephadex G-25 column to recover
16 mg of a CHM-conjugated peroxidase.
21 mg of the aforementioned reduced complement
component Clq and 14 mg of the CHM-conjugated peroxidase
were mixed together, and the mixture was allowed to stand
stationarily a-t 4~ to 10 C for 15 hours and then passed
through a Sepharose 6B ~Trade Name-of Pharmacia Fine Chemicals
Co.) column to recover a fraction of a molecular weight range
of from 400,000 to 800,000 to obtain 29 mg of a complement
component Clq labelled with peroxidase.
Example 2
En~yme-conjugated Complement Component Clq:
1 mg of ~-D-galactosidase derived from Escharichia
coli was dissolved in 0.2 mQ of a O.lM phosphate buffer(pH 6.0),
and then reacted with 0.1 mg of N,N'-o-phenylenedimaleimide
dissolved in 0.2mQ of a phosphate buffer containing 5X
dimethylformamide at 30~ for 25 minutes. The solution
containing the reaction product was passed through a Sephadex
f r ~ ~l e n~
G-25 ~r~ee-~hu:~ of Pharmacia Fine Chemicals Co.) column
equilibrated with a phosphate buffer containing 0.2 mg/mQ of
bovine serum albumins, whereby a 720 ~ g of ~-D-galactosidase
coupled with maleimide.
- 31 -
::
, .~, .. ,: . . . . .
.- .. .
. .
2~0 ~ g of the ~-D-galactosidase coupled with
maleimide ~as dissolved in 0.1 mQ of a phospha-te buffer
containing 1 mM of sodium ethylenediamine tetra-acetate, and
reacted with 2 m~ of the recuced comF~lement componen-t Clq
prepared in Example 1 and dissolved in 0.1 mQ of a phospha-te
buffer containi~g 1 mM of sodium e$hylenediamine tetra-acetate
at 4C for 48 hours. The solution containing -the reaction
product was subjec-ted to gel filtration usin~ a SePharose 6B
tt~a t e n~ r/~C
~T~a~ ~a~ of Pharmacia Fine Chemicals Co.) column, and then
processed -through the procedures as described in Example 1 to
obtain an active fraction, i.e. a fraction containing 1.6 mg
of a complement component Clq combined with ~-D-ealactosidase.
Example 3
En~yme-conjugated Complement Component Clq:
~1) Preparation;
Generally following to the same procedures as in
Example 1, except that a goat serum was used in place of the
rabbit serum, a reduced complement component Clq was prepared,
which was then conjugated with peroxidase to obtain a
peroxidase-labelled complemen-t component Clq.
(2) Test;
Using a serum having a CF antibody titer of 1~, the
reactions of the thus obtained pero~idase-labelled complement
component Clq with a herpes simplex virus CF antigen and with a
- 32 -
: ,~
.. . ... . . .
-
..
- -
-- , .~ ,
~2~8~8
normal cell antigen were inspected ~y means of the solid phase
en~yme immunoassay to obtain the results as set forth in the
following Table 1. In Table 1, the results of this Example are
shown together ~ith the results of the following Comparative
Example 1.
Compara-tive Example
Enzyme-conjugated Complement Component Clq Prepared by
Conventional Process and Having the Enzyme Coniugated
~enerally at Random:
(1) Preparstion;
1.5 mg of horseradish peroxidase ~as dissolved in
0.2 mQ of distilled water, and added with 60 ~ Q of a O.lM
sodium periodate solution, followed by agitation at room
temperature for 20 minutes. The solution was dialized through
an acetate buffer (pH 4.4) containing lM sodium chloride, added
~ith 60 mg of sucrose, and then addecl with 1 mQ of a carbonate
buffer (pH 9.2) containing 3 mg of purified goat complement
component Clq and also containing lM sodium chloride and 10~ of
sucrose. After agitating for 2 hours, the mixture ~as further
added with 0.1 m Q of a 4 mg/m ~ solution of sodium borohydride9
and then allo~ed to stand at 4C for additional 2 hours.
Thereafter~ the admixture was subiected to gel filtration
7~q~evnq ~ti
through a Sephacryl S-300 ~p~e-~4~r~ of Pharmacia Fine
Chemicals Co.) column, and the fraction having both of the
- 33 -
~ _ i
,.. ,. ,...... , - -, .- . :
.. , : . :
: . '-.' : '' :`' ~
~2~41~3
peroxidase activity and the Clq activity was collected.
(.~) T _ ;
Using a serum having a CF antibody ti-ter of 1~, the
reactions of the thus obtained peroxidase-labelled complement
component Clq with a herpes simplex virus CF antigen and with a
normal cell antigen were inspected by means of the solid phase
enzyme immunoassay to obtain the results as set forth in the
following Table 1. In Table 1, the results of this Comparative
Example are shown while comparing with the results of Example 3.
Table
.
Color Development Color Development
of Herpes CF of Nor~al
Antigen Well Antigen Well
Enzyme-Labelled Clq 0.544 0.07
of Example 3
Enzyme-Labelled Clq 0.328 0.281
of Comparative Example 1
It should be appreciated from the results shown in
Table 1 that the enzyme-labelled complement component Clq
prepared by the conventional process reacts with the normal
antigen inselectively or non-uniquely and and has a low or
feeble capability of reacting wi-th the herpes simplex virus CF
antigen selec-tivel~ or uniquely; whereas the enzyme-labelled
- 34 -
.. .~
.
,
~26~34~8
complement component Clq prepared by the process of the
invention is considerably lowered in inselectlYe or non-unique
reaction wi-th the normal antigen to have a su-fficiently high
capability of reaction with the herpes simplex virus CF antigen.
Example 4
Toxin~Coniuga-ted Complement Component Clq:
(1) Preparation;
4mg oE purified ricin A chain was disolved in 1.2 mQ
of a phosphate buffer (pH 7.0) containing 20~ of dimethylform-
amide, and added with 30 ~ Q of a phosphate buffer containing3~ of 4-(maleimideme-thyl~cyclohexane-1-carboxylic acid
succineimide ester (hereinafter referred to as CHM) to react
at room temperature for an hour. Then, the solution containing
the reaction product was passed through a Sephadex G-25 column
to obtain 2.3 mg of a CHM-conjugated ricin which was dissolved
in 1 m ~ of O.lM phosphate buffer (pH 6.0) and then added with
2.5 mg of the reduced complement component Clq prepared by the
same process as in Fxample 3 and dissolved in 0.5 mQ of a
phosphate buffer to react with the latter by allowin~ to stand
the mix-ture at 4&~ for 22 hours. The reaction mixture was
~T~q4~
subjected to gel filtration using Sephacryl S-200 (~r~ e
of Pharmacia Fine Chemicals Co.) to obtain l.9 mg o-f a
ricin-conjugated complement component Clq.
(2) Test;
- 35 -
: ::: ~ : ~: ,, , :
lX~8~18
The T cell was refined from -the BALB/c mouse spleen
cell primed with DNP-KLH by passin~ the primed cell through a
Nylon wcol column. The T cell -fraction was put into a
RPI~I-16~0-10~FCS culture medium containing anti-mouse Ly-2,3
antiserum and 2 ~ g /mQ of the aforementioned ricin-conjugated
complement component Clq, and then allo~ed to stand stationarily
at 37~C for an hour. Thereaf-ter, the processed cell ~as rinsed
with a Hanks' balanced salt solution, cultivated in a
RPMI-1640-lO~FC~ culture medium containing mouse Interleukin 2
for 7 days, and the distribution of the recovered cell Ly
antigen was checked to find that the number of cells havin~
the Ly-1 antigen on the surfaces thereof were increased as
large as 1.6 times of those of a control which had not been
processed with the anti-mouse Ly-2,3 antiserum and the
ricin-conjugated complement component Clq.
Example 5
Dyestuff-Coniugated Complement Component Clq:
(1) Preparation;
10 mg of purified bovine serum albumin was dissolved
in 1 mQ of 0~5M carbonate buffer and added with 0.4 mg of
fluorescein isothiocyanate (hereinafter referred to as FITC).
After reacting the mixture for 7 hours, the reaction mixture
was subjected -to gel filtration to obtain bovine serum albumin
combined with FITC. 8.2 mg of the bovine serum albumin
- 36 -
.~.. .
....
.
- . -: ' .
. ., - -
~L~6 ~ L~3
combined with FITC was then dissolved in 0.4 m~ of a O.lM
sodium phosphate buffer (pH 7.0), and added wi-th 50 ~ Q of
a 90 mg/m~ solution of CHM in dimethylformamide -for reaction
at 30DC for an hour. After removing the insoluble materials
by cen-trifugal separation, the buffer solution was exchanged to
a O.lM phosphate buffer ~pH 6.0). 0.56 m~ of the solution
was added with 0.5 m Q of a O.lM phosphate buffer (pH 6.0)
containing 8 mg of the reduced complement component Clq prepared
in Example 1 and 5 mM sodium ethylenedia~ine tetra-ac0tate, and
the admixture was maintained at 4~ for 18 hours. After then,
the admixture was filtered through a gel filter of Sepharose 6B
column to obtain 12 m Q of an eluate fraction having a molecular
weight ranging wi-thin 400,000 to 900,000 and containing a
reaction product between the complement component Clq and the
bovine serum albumin combined with the FITC.
(2) Test
Separately, the spleen cells ~ere removed from the
BDF1 mouse and passed through a Nylon column to obtain T-cells.
The thus obtained T-cells, a rabbit antiserum against mouse
brain associated T-cell antigen and the eluate fraction prepared
through the process desribed in the preceding paragraph were
mixed together to form a mixture having a concentration of
150 times of the final concentration. After maintaining the
mixture on ice for an hour and rinsing sufficiently, the cell
- 37 -
:
, . . : ~,
, : :. : , - .. .. . ....
. . . . . .. . . ' .
~L2~8418
was floated on -the surface of a 50X glycerin-phosphate buffer
solution and observed through a fluorescent microscope. The
resul-t was that 93~ of the cells emitted fluorescent light to
reveal that almost all of the cells were the T-cells.
Example 6
Complement-Component Clq Coniugated with ~onor or Acceptor
for Electron:
(1) Preparation;
Chlorophyllin a was dissolved in distilled water so
that a 1 mg/mQ solution was formed, and the pH value of the
solution was adiusted with hydrochloric acid to pH 7.5, ~ollowed
by addition of 1-ethyl-3-(3-dimethylaminopropyl~carbodiimide
chloride and ethylenediamine so that the resultant reaction
mixture contained O.lM of the former and 0.8~ of the latter.
The mixture was allowed to stand for reaction for 120 minutes.
The reaction product was purified by the use of CM-Sephadex
Je ~
(Tr~dc Name-of Pharmacia Fine Chemicals Co.), and 450 ~ g of
aminoethylated chlorophyllin a was dissolved in 0.5 m Q of a
O.lM phosphate buffer (pH 7.0) containing 40~ of dimethyl-
formamide. The solution of puri-fied aminoethylated
chlorophyllin a was mixed with Q.S m Q of a 40~ dimethyl-
formamide solution containing 2~ of GHM, and maintained at
30~ for an hour to react with CHM. The reaction mixture was
Tr o~ ~ a ~
subjected to gel filtration using Biogel P-2 (~ade-Na~e of
. ~ , . ,. ~.
, , - ,: .; ~ , . . .
:~
~L268418
Bio-Rad Laboratories Inc.) -to obtain CHM-chrolophyllin a.
12 m Q of the reduced complement component Clq p~epared in
accordance with the process as described in Example 3 and 1~
~ g o~ the CHM-chrolophYllin a ~ere put into 1.5 m Q of a O.lM
phosphate bu-ffer (pH 6.0), and allowed to stand stationarily
at 4C for 18 hous~ followed by gel filtration at which a
fraction having a molecular weight ranging within 400,000 to
600,000 ~as picked up, whereby 7.5 mg of chrolophyllin-labelled
complement component Clq was obtained.
(2) T
Separately, a chemically modified antigen electrode
was prepared by coating an antigen protein against herpes
simplex virus on an SnO~ Nesa electrode so that the antigen
protein was combined with the electrode through a covalent bond.
The electrode ~as immersed in a 25 mM phosphate buffer (pH 6.9~)
containing 50 mM hydroquinone, a 1/50 final concentration of a
human blood serum (CF = 32) including an anti-herpes simplex
virus, and 10 ~ g of the chrolophyllin-labelled complemen-t
component Clq, and irradiated by a white light while maintaining
2a the electrode potential at 0.1 Vs- SCE, ~hereupon genera-tion of
pho-tocurrents was observed. The quantum efficiencies of
photocurrents were about 9~.
Example 7
Magnetizable Substance-Coniugated Complement Component Clq:
.
- 39 -
` ~
- ..... ..... . .. ..... ...
.: .,. ., ~
.- ~ :,. - ; ~ :
~268~1~
(1) _epara-tion;
Polystyrene microbeads each having amino groups at
the surface thereof and containing micro grains of magnetite
were suspended in 1 mQ of O.lM phosphate buffer (pH 7.0)
containing 20~ dimethylformamide, and added with 40 ~ Q of
O.lM phosphate buffer containing 2.5~ CHM, followed by moderate
agitation at 30~C for 60 minutes for reaction. After
rinsing the beads, they were suspended again in 1 mQ of O.lM
phosphate buffer (pH 6.0) and added with 0~5 mQ of another
phosphate buffer containing 1.8 mg of the reduced complement
component Clq prepared by the process as described in Example 3,
and then the admixture was allowed to react at 4C for 18 hours
under moderate agitation. The beads ~ere rinsed with a Veronal
buffer solution (pH 7.4) containing 0.1~ of gelatin, the
solution being referred to as ~VB hereinafter, and then stored
in the ~VB at 4C.
(2) Test;
Separately, the spleen cells of X5563 tumor-bearing
C3H/He mouse were cultivated on a culture medium containing
IL-2, and the once rinsed cells were again floated on the same
culture medium and mixed with the beads-coniugated complement
component Clq prepared by the process described in the preceding
paragraph and an anti I-Jk antiserum, followed by stationary
standing at 37C for 2 hours. After the lapse of the pre-set
- 40 -
, ~ . .
,`:' :'
~L~;8 4~L~3
-time, the cells were collected and then the collected cells
were again floated gently, and an intense magnetical force
was applied from -the ex-terior of the container to capture the
cells having the I-Jk antigens at the surfaces thereof. The
cytotoxic activity of the cell left in the culture mediu~
against the X5563 tumor cell was recognized to be about 1.4
times higher -than -that of -the cell cultivated for 5 days in a
simple IL-2.
Example 8
Measurement of Complement-Binding Antibody:
On a 96 ~ell microtiter plate absorbing a complement-
fixed antigen of the herpes simplex virus added, respectively,
were 5 ~ Q of each of sample sera inactivated to have
complement-binding titers of less than 4 and 1~, and then 95
Q of the peroxidase-labelled complement component Clq in
tr~den~
Example 1 (diluted to 100 times volume ~ith a gelatine-Veronal
buffer solution~ was added, followed by stationary standin~ at
room temperature for an hour, Thereafter, each well was rinsed
three times with a phosphate buffer containing O.Q5X of Tween-ZO
~a. ~ral~ar~K ~cO~
~ (a surface active agent produced and sold by Nakarai Chemicals
LTD.), and added with 100 ~ Q of an HzOz-hBTS solution [2,2'-
adino-di-(3-ethyl-benzothiazoline sulfate) solution containing
hydrogen peroxide], followed by standing at room temperature for
an hour to complete the reaction. After adding with lOQ ~ Q of
- 41 -
, . . .
~2~i84~8
an en~ymatic reaction terminating agent, the light absorbance
at 414 nm was measured to -find that the absorbance of the serum
having the complement-binding titer o-f less than 4 was 0.029
~hile that of the serum having the complement-binding -titer of
16 was 0.579.
Example 9
Measurement of Complement-binding Antibody:
Bovine blood serum albumin was dissolved in a
saline solution buffered by phosphate to prepare a solution
having a concentration of 20 ~ ~/mQ , which was poured in
each well of a 96 well microtiter plate and then maintained
at room temperature for 2 hours to be absorbed by each well.
After removing free bovine blood serum albumin, 50 ~ Q of
anti-bovine serum albumin rabbit an-tiserum ~stepwisely diluted
by 800 to 6400 times with a gelatin-Yeronal buffer) and 50 ~ Q
of the ~-D-galactosidase -labelled complement component Clq
synthesized in Example 2 were added, and the microtiter plate
was allowed to stand at room temperature for 3Q minutes. After
rinsing each well, 100 ~ Q of o-nitrophenyl ~-D-galactoside
solution (in a phosphate buffer solution having a pH value of
7.3) was added, followed by standing at room tempereture for 60
minutes, and then 0.1 mQ of a O.lM sodium carbonate solution
to terminate or cease the enzymatic reaction. The light
absorbances of respective wells were measured at a wavelength
- ~2 -
, ,
: ~ , . ,.. .. . :
:~Xt~411~
of 420 nm to find that the ligh-t absorbances were graduallY
varied from 0.421 to 0.063 depending on the change in dosed
amounts of antiserum.
Example 10
Measurement of Complement-binding Antibody:
A purified antigen of herpes simplex virus was
absorbed by 6.35 mm of polystyrne beads which were put into a
small test tube, and added simultaneously with 0.1 m~ of a 10
times diluted solution of a solution of each of inactivated test
sera (having the complement-binding ti-ters of 16 and less than 4)
in a gelatine-Yeronal buffer and with 0.1 mQ of a solution of
the peroxidase-labelled complement component Clq prepared in
Example 1 diluted with the same buffer. The admixture was
then allowed to stand stationarily at room temperature for
an hour. After rinsing the beads, they were transferred to
another small test tube, and added with 0.3 m~ of an
o-phenylenediamine solution to react at room temperature for
45 minutes. The reaction was terminated by the addition of
2 m e of 1 N hydrochloric acid, and the light absorbances of
the samples at the wavelength of 490 nm were measured. The
sample having the complement-binding titer of less than 4
had a light absorbance of 0.018, whereas the sample having
the complement-binding titer of 16 had a light absorbance of
0.408.
- 43 -
. .,
: . ':, '' :. ~ . : :
1~84~
E~ample 11
Measuremen-t of ~ntigen:
A 96 ~ell micro-titer plate absorbing guinea pi~
anti-herpes simplex virus antibody (Fab) was supplied wi-th O.l
m~ of uterus cervix swabs of a patient, and stationarily held
at room tempera-ture for 60 minu-tes. After rinsing the pla-te
wi-th the PBS ~a 0.85g saline-containi;ng phosphate buffer having
a pH value of 7.4) -for three times, each well ~as added with
0.05mQ of ei-ther one of the inactivated guinea pig anti-herpes
simplex virus sera (having the complement-bindin~ antibody
titers of 16 to 32) and also with 0.05mQ of the peroxidase-
labelled complement component Clq prepared in Example 1. The
plate was held stationarily a-t room temperature for 60 minutes.
Then, each well was rinsed with PBS containing 0~05~ of T~een-20
~r~l temQ r~/~
15 ~ (a surface active agent produced and sold under such ~ ae
~la~ from Nakarai Chemicals LTD.), and added ~ith 0.01 mQ of
H20z-ABTS [2,2'-adino-di~3-ethyl-benzothiazolin sulfate)
containing hydro~en peroxide] solution to be held at roQm
temperature for an hour for reaction. Thereafter, 0.05 m~ of a
0.05~ aqueous sodium nitride ~hich acted to terminate the
enzymatic reaction, and then the light absorbances of respective
sample wells a-t a wavelength of 414 nm were measured to find
that the sample well filled with uterus cervix swabs of a
patient who was nega-tive against the herpes simplex virus had an
- ~4 -
:. .:
~68418
absorbance o-f 0.030 and -that the sample wells filled with uterus
cervix swabs of a patien-t who were posi-tive against the herpes
simplex virus had absorbances of 0.1139 O.~OOs 0.550 and so on.
E~ample 12
Measurement of Antigen:
According to a conven-tional process, a lymphocy-te
fraction was prepared from the mouse spleen cell, followed by
rinsing ~ith thc PBS, and then the concentration of the cell
was adjusted to l X 10~/mQ . 0,1 mQ o-f the thus prepared
lYmphocyte fraction, 0.05 mQ of antimouse Thy-1,3 alloserum,
and 0.05 m Q of -the FITC-labelled complement component Clq of
Example 5 ~ere mixed together and allowed to stand at room
temperature for an hour. The cells were then rinsed thoroughly
with the PBS and observed through a fluorescent microscope.
The result revealed that 37~ of the cells were fluorescent.
Example 13
Measurement of Antigen:
The lymphocytes in a blood of a leukemia
patient were suspended in 1 mQ of a phosphate buffer to
prepare a suspension containing 1 X 108/mQ of lymphocytes,
and the suspension was processed by an ultra-sonicator for
2 minutes. The homogenate ~as then clarified by centrifugal
separation, and the supernatant was added ~ith 0.5mQ of DNA
Sepharose to react -therewith at 37C for 60 minutes. The DNA
45 ~
,. ~; , . ...
..
~2~84~8
Sepharose was bound with DNA rela-ted enzymes, such as DNA
polymerase and terminal deoxynucleotidyl transferase (TdT).
Then, O.l m Q of the peroxidase-labelled complement
component Clq and an inactivated rab~it anti-TdT serum,
followed by reaction at 37C for 30 minutes. After rinsing
thoroughly with PBS, the DNA Sepharose was recovered, to which
1 mQ of a solution of HzOz-ABTS, was added, and the admixture
was reacted at 37~C for 60 minutes. Then, 1 mQ of a 0.05~
aqueous solution of sodium nitride acting as a terminator for
the reaction, and the light absorbance of the supernatant was
measured at a ~avelength of 41~ nm. It could be iudged that
the sample having a light absorbance value of not more than
0.075 showed that the patient was nega-tive to TdT and that
the sampla having a ligh-t absorbance value of not less than
0.100 showed that the patient uas positive to TdT and suffered
from acu-te leukemia.
Example 14
Measurement of Neutralizing An-tibodY:
Two sample sera having, respectively, neutralizing
antibody titers of 32 and 128 to the HSV (Herpes Simplex Virus)
~ere diluted with a phosphate buffer to have the volumes four
times as lar~e as the initial volumes, heated at 56C for 30
minutes -to be inactivated, and then further diluted with the
same buffer to have e~iht times volumes. O.l mQ for each of
46 -
~: . ; ,,. : ~
lX~8~
-the -thus inactivated and diluted sample sera was mixed with
0.7 mQ of the same buffer containing 4 X 103 pfu/mQ of HSV,
and then kept at 37C for 60 minutes to proceed the reaction.
Separately, Vero cells had been cultivated through the
S momolayer culture on a microplate, onto which a mixture of the
serum and the HSV was added at a content of 50 ~ Q /well, and
then the virus was absorbed by holding the plate in a culture
filled with 0.5~ carbon dioxide and maintained at 37C for 60
minutes, with the addition of a maintenance medium followed by
cultivation for additional 24 hours. Then~ the cell was fixed
by the use of me-thanol containing 3~ of hydrogen peroxide.
After fixing by the methanol containing 3~ of hydrogen
peroxide, as described in the preceding paragraph, 50 ~ Q
for eaGh of human sera having complement-fixing titers against
the HSV diluted by 50 times ~ith a gelatine-Veronal buffer
(pH 7.4), respectively, of 16 and less than 4 was poured
into individual wells, and then each well was added with
0.2 ~ g/50 ~ Q /well of the peroxidase-labelled goat complement
componen-t ~lq prepared in Example 3 and dissolved in the same
; 20 buffer. After allo~ing to stand the microplate at room
temperature for 2 hours, each well was rinsed with a phosphate
buffer solution containing 0.05~ of Tween 20 ~or three times,
and then added with 0.7 mQ /well of a Hz08-ABTS solution to
develop coloring of each well which was subjected to light
47
~'
~L~t;~34 1 8
absorbance de-termination conducted at a wavelength of 419 nm.
The results are shown in Table 2.
Table 2
Serum for Detec-tion OD414
of Residing Virus (1) - (23
. . . _
Serum for Well (1) Well (2) Added
Determination of Added with wi-th Normal
Presnece or Absence ofGuinea PigGuinea Pig
Neutralizing AntibodyAnti-HSV Serum Serum
Control (Well Not Added0.618 0.095 0.523
with Sample Serum~
..................................................... ............................. ............................. ..................
Sample Serum Having Neutra 0.504 0.092 0.412
lizing Titer of 32
..................................................... ............................. ............................. ..................
Sample Serum Having Neu-tra 0.117 0~101 0.016
lizing Titer of 128
15 Example 15
Measurement of Neutralizing Antibody:
An anti-HSV positive human blood serum having a
neutralizing antibody titer of 1~8 and a negative human blood
serum ~ere diluted by four times with a phosphate buffer, and
inactiva-ted, and then a serial dilu-tion series diluted by
4 to 512 times was prepared each for the bo-th sera on a
micro-ti-ter plate provided with a number of wells each having
a volume of 0.1 mQ /well. Each well was filled with O.lmQ
of a buffer containing 4 X 103 pfu/mQ of HSV. The following
- 48 -
,, "
-, :,. -: ,, :
- . ~- , ; -,
~26~34~8
procedures were the same as in Example 14 -to measure or
determine the OD414. The results are plotted in the graph
illustrated in Fig.1 wherein the abscissa indicates the
dilu-tion rate of each serum and the ordinate indicate
the OD414. As shown, for the positive serum, -the dilution
rate giving the value as large as lt2 of the maxi~um OD414
corresponds to the neu-trali~ing antibody titer of 128.
E~ample 16
Measurement of Intracellular Substance or Microorganism:
A specimen to be inspected was picked up -from a
defected portions of HSV infected patient ~pendedum or labia),
and suspended in 1 mQ of culture medium solution containing an
antibiotic. 0.1 mQ of the suspension was inoculated -to two
wells of a microplate in which Yero cells had been preliminarily
cultivated, and further cultivated at 37C for 22 hours.
After the completion of 22 hour cu1-tivation, the
cultivated cells ~ere fixed with 3~ hydrogen peroxide-methanol,
and 50 ~ Q of a human serum diluted by 25 tmmes with a
; gelatine-Veronal buffer (pH 7.4),the serum having an anti-HSV
complement binding titer (CF titer) of 32 or less than 4, was
put into individual wells together with 50 ~ Q of a solution
in the same buffer containing 90 ng of the peroxidase-labelled
complement component Clq prepared in Example 3. hfter reacting
at room temperature for 2 hours, each well was rinsed with a
- 49 -
',; ., ", ''.
841~
phosphate buffer containing 0.05~ of Tween 20 for three times,
added wi-th O.lmQ /well of a hydrogen peroxide-ABTS solu-tion
(pH 4) followed by standing for an hour to develop coloring, and
then -the reac-tion was terminated by the addition of 0.1 mQ of a
0.01~ sodium azide. Thereafter, the light absorbance of the
reaction product in each well was measured. The well added
with the human blood serum having a CF titer of 32 had a light
absorbance of 0.263, whereas the well added with the serum
having a CF -titer of less than ~ had a light absorbance of
0.089. From those result, it could be confirmed tha-t the HSV
virion was present in the specimen inspected.
Example 17
~easurement of Intracellular Substance or Microor~anism:
With the aim -to cloning a cell producing carcino
embryonic antigen, the cell T3~-4 producing CEA from pancreas
tumor was diluted to the limit (i.e. to 1 cell/well)1 and then
cultivated on a 96 well microplate for 16 days. After removing
the culture medium solution~ 0.1 mQ of tripsin~sodium
ethylenediamine tetra-acetate was put into each well to float
2~ the cells, and then two plates preliminarily filled with 0.2 m Q
/well of a fresh culture medium solution were replicated so that
repricas containin~ 20~ Q /well of floating cells were prepared.
The culture medium solution in one of the repricas was thrown
away, followed by fixation of the cells wmth 3~H2Qz-methanol,
- 50 -
. . .
: ' ' : .,' , " .,
.
8 4 1~3
and then added with 0.1 mQ of a 1/400 time diluted rabbit anti-
CEA antiserum diluted ~ith GVB (gelatine-Veronal buffer) and llO
ng/O.lmQ /well of the peroxidase-labelled complement componen-t
Clq. After reacting at room temperature for 2 hours and rinsing,
coloring of -the well was developed b~l the addition of a solution
of the substrate of ABTS ~diammonium ~2,2-azi)~di~3-ethylbenzo-
thiazolin sulfonic acid)]. The OD4l4 of respective wells ranged
within 0.127 to 0.386. The cell in the well showing the maximum
OD4l~ was picked up from -the other reprica, and subjected to
expansion.
Example 18
Measurement of In-tracellular Substance or Microorganism:
100 ~ g ~0.1 mQ ) of purified ~-fetoprotein and
O.l mQ of Freund complete adiuvant were mixed together and
dosed into the abdorminal cavity of a 7 week age Blb/C mouse.
After 28 days from the dosage of the aforementioned materials,
100 ~ g (0.3 mQ ) of of AFP (~-fetoprotein) was additionally
dosed, and after 3 days of -the dosage of the AFP, the renal
cells were picksd up and fused with NS-1 cells. The cells
were spread over a 96 well plate at a concentration or
distribution density of l X lDs/mQ . From the first day to
the sixteenth day after fusing, selection by the HAT medium
(hypoxantine-thy~idine-aminopterine medium) was conducted,
and the antibody activity of the supernatant of each cultivated
- 51 -
~L;~ti8~L1~3
well was inspected on -the seven-teenth day to reveal -that
prodcu-tion of antibody was recognized at a rate of 64/9~8 wells
and prodcution of anti-AFP antibody was recognized in two wells.
The cells in respective wells were utilized as coated specimens
while being processed by 3%HzO~-me-thanol to be fixed, and then
add~d with 20~ e of a goa-t anti-mouse IgG (~ -chain selectivi-ty)
diluted by 200 times and also wi-th 18 ng/20~ ~ of the
peroxidase-labelled complement component Clq, followed by
stational standing for 2 hours. After rinsing -thoroughly,
development of coloring of each cell was effected in a
diaminobenzidine solution, and the number of cells producing
the IgG was counted to find that the ratio of positive cells
l~ere 62~ and 9l~, respectively.
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