CA1268418A - Substance-conjugated complement component c1q - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
A substance-conjugated complement component Clq is provided. A substance such as signal emitting substances or cell function regulating substances is conjugated via a sulfur atom to at least one site of the component. The site is not involved in binding immunoglobulins. A marker-labelled complement component Clq is used for measuring a complement-binding antibody, an antigen, a neutralizing antibody or a substance produced internally of and at the surface of a cell or a microorganism by measuring the marker.

Description

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 ~ "' ~ '', ' .~, , .

.,- . , - , . .
" ... . .

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

- 2 -, "

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 : ~
` - 3 -~, .
~ - , :' - - : : ~
- . . - , - ., .

: , : : . , .:: ~ ,. :

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 ' . ' ~ ` ` ' ' '~
`
'' ` ' . . ' . ,'. `
` . ` ' ' '' ' ""'` ~ . :`

~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 ;
`~-' 5 .".' , , ~.... . . . .

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:

:

.. . . . . .
. . - , . ~ , . .

~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 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 : - 8 -;, . . .

- : . ' .. ' ~ ~ -: - - . .. :,: - :

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 - g ` ' ' ""' ' ' "' ' ~ ' -: , . .
' : :' 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 ' :. .
' . ', ''. . ~
:

~26~ 8 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 . _ ., . ~ .

~' ' " .. ,:
. '.~'. ;, ' '' .

.,-.~ ,. ~ '. . . . . .

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 .~ ~ :., ., ,:

: , ; .. ~ - . , -~L~6~4~

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 ., _ . ~ . , . , ~
,' ,,~ ~ . ' ;`i' ~ , ' ''. ' ~'' ` ~ ' ~L~ 3~3L8 -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, ' ~

...... .. . .. - : : . -, .. , ~ ~ :. .-- , : . :- ,, . : , : . ~ .
.;: .,. "..... : . , , . ., ~ , . , :
-: :. ~:, . .
.: ~ . . ..
.

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 - ~6 -" _ .

~,; . - ~ ~ - - ;
, :; -~L2~3~3L~8 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 ,; . , .: . . , 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 : `. :

. ~

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, ,. '' '.' .~,; ,: ,;, , " ' ~
. ~ . : .

, .
.

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 .

: . ~

~ . , , ~ . , . :

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 ,,.~.

.:", . . ..

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 : :

,~.. : , :....... ~.

: :.. , :.

~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 :

,,". ., - -:

~ .: . ~ ,. ; .

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.

,,.. , , . . , -.- . . ..

- . :, -, ~

:: - ~ , . .

~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 . . .
.
. .
;- . . , .
... ~: .

~;26~3418 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-. .

- . , . ;., . , . :
- :.~ , . . . .
- - . . -. ,. ~ . ... ,~, . .
. , .. , . ~- :

~: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 : ; -: .
- . " .
.. .

., . ' ' '. "'' ' . ,'. ., ~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.

.

.. - :. , .
" : : ..
- -. ~ . ,.
: ~ . , . .... :

~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, .

: "; , :
~ ~ : -, -, '' - ,: `' .. .

, ~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.

::
, .~, .. ,: . . . . .

.- .. .
. .

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 : ,~

.. . ... . . .
-..

- --- , .~ , ~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 ~ _ 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 .. .~

.
, ~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;

: ::: ~ : ~: ,, , :

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 .~.. .

....
.

- . -: ' .

. ., - -~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 :

, . . : ~, , : :. : , - .. .. . ....

. . . . . .. . . ' .

~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:

.

` ~
- ..... ..... . .. ..... ...

.: .,. ., ~
.- ~ :,. - ; ~ :

~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 , ~ . .
,`:' :' ~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 , . . .

~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.

. ., : . ':, '' :. ~ . : :

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 ~: . ; ,,. : ~

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 ~' ~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 ,, "
-, :,. -: ,, :

- . ~- , ; -, ~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 ',; ., ", ''.

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, . . .

: ' ' : .,' , " ., .

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 ~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.

.. . .. .

.. ..
. . . -- ,~ .

Claims (33)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A complement 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.

2. The complement component Clq according to claim 1, wherein said substance is selected from the group consisting of signal emitting substances and cell function regulating substances.

3. The complement component Clq according to claim 2, wherein said signal emitting substance is selected from the group consisting of enzymes, coenzymes, enzyme substrates, dyestuffs, magnetizable substances, donors and acceptors for electron transference, radioactive substances, metal compounds, and metal compositions.

4. The complement component Clq according to claim 3, wherein said enzyme is selected from the group consisting of peroxidases, alkaline phosphotases, galactosidases and alcohol dehydrogenases.

5. The complement component Clq according to claim 3, wherein said coenzyme is selected from the group consisting of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide and flavin adenine dinucleotide phosphate.

6. The complement component Clq according to claim 3, wherein said enzyme substrate is selected from the group consisting of o-nitrophenyl-.beta.-D-galactopyranoside and 3-hydroxysteroid.

7. The complement component Clq according to claim 3, wherein said dyestuff is selected from the group consisting of methylene blue and fluorescein isothiocyanate.

8. The complement component Clq according to claim 3, wherein said magnetizable susbstance is selected from the group consisting of carbonic iron, iron-containing microcapsules and complexes of iron with proteins.

9. The complement component Clq according to claim 3, wherein said donor and acceptor for electron transference are chlorophyl.

10. The complement component Clq according to claim 3, wherein said radioactive substance is selected from the group consisting of 124 I-labelled albumin, p-chloro(203Hg)mercuri-benzoic acid, N-ethyl(2,3-14C)maleimide and iode(l-14C)acetamide.

11. The complement component Clq according to claim 3, wherein said metal compound and said metal composition are selected from the group consisting of gold colloid and iron-containing microbeads.

12. The complement component Clq according to claim 2, wherein said cell function regulating substance is selected from the group consisting of antibiotics, growth factors, hormones, cell activation factors, cell division factors, anticancer agents and toxins.

13. The complement component Clq according to claim 12, wherein said antibiotic is selected from the group consisting of amphotericin B and actinomycin D.

14. The complement component Clq according to claim 12, wherein said growth factor is selected from the group consisting of selenium compounds, insulin, transferrin and epidermal growth factor.

15. The complement component Clq according to claim 12, sherein said hormone is corticosteroid.

16. The complement component Clq according to claim 12, wherein said cell activation factor is a macrophage activation factor.

17. The complement component Clq according to claim 12, wherein said cell division factor is a B cell division factor.

18. The complement component Clq according to claim 12, wherein said anticancer agent is mitomycin C.

19. The complement component Clq according to claim 12, wherein said toxin is ricin of toxin of castor bean.

20. A process for preparing a substance-conjugated complement component Clq, comprising the steps of:
(a) adding a reducing agent to a complement component Clq to cleave at least one S-S bond present at a site not involved in binding immunoglobulins thereby to obtain a reduced complement component Clq having at least one exposed -SH
group; and (b) conjugating a substance to said complement component Clq via said exposed -SH group.

21. The process according to claim 20, wherein said reducing agent is a sulfur-containing compound.

22. The process according to claim 21, wherein said reducing agent is selected from the group consisting of mercaptoethylamine, dithiothreitol, 2-mercaptoethanol, cysteine and glutathione.

23. The process according to claim 20, wherein said reducing agent is allowed to act on the complement component Clq which is dissolved in a buffer solution for permitting the complement component Clq to be present stably.

24. The process according to claim 20, wherein said buffer solution is selected from the group consisting of tris buffered saline and phosphate buffered saline.

25. The process according to claim 20, wherein said reducing agent is allowed to act on said complement component Clq at -2°C to 45°C for 30 seconds to 24 hours.

26. The process according to claim 20, further comprising a step of removing excess reducing agent after the completion of said step (a).

27. The process according to claim 20, wherein said substance is selected from the group consisting of signal emitting substances and cell function regulating substances.

28. The process according to claim 20, wherein said substance is conjugated directly to said exposed -SH group.

29. The process according to claim 20, wherein said substance is conjugated to said exposed -SH group indirectly via a second susbstance having coupling function.

30. The process according to claim 20, wherein said second substance having said coupling function has a group selected from the group consisting of maleimide residue and -SH group, and also has a group for coupling said substance.

31. The process according to claim 20, wherein said substance is peroxidase extracted from horseradish, and wherein said second substance having said coupling function is N-hydroxysuccinimide ester of maleimide.

32. The process according to claim 20, wherein said substance is conjugated to said reduced complement component Clq in a buffer solution for both of said substance to be conjugated and said reduced complement component Clq.

33. The process according to claim 20 wherein said step (b) is a step of conjugating said substance indirectly via a second susbstance having coupling function and via said exposed -SH group to said complement component Clq, and wherein said step (b) is effected in a common buffer for said reduced complement component Clq, said second substance having coupling function and said substance to be conjugated.