CA1276103C - Substance-conjugated complement component c1q - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
A method for measurement using a substance-conjugated complement component Clq is provided. A substance such as signal emitting 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

~271~3 TITLE OF TllE INVENTI~N:
Subs-tance-conju~ated Complement Componen-t Clq BACKGR~UND OF THE INVENTION:
Field of the Invention;
This invention relates -to a method for measurement, using a substance-conjuga-ted complement component Clq conjugated with various substances including markers.
Related Art S-tatemen-t;
It. has hitherto been known to utilize the complemen-t fixation reaction for the measurement or determination of antibodies in blood serum and antigens, such as microorganisms, phisiologically active substances and chemicals. This known method makes use of the serial reactions wherein complement components Cl to C9 are bound successively to an antibody specifically bound to an antigen. In detail, this known method comprises the step of adding an excess amount of complement components to the formed antigen-antibody complex~ the step of determining an amount o-f residual complement components through the hemolysis, and the step of de-termining the amount of ~O fixed complement components from the degree of hemolysis.
The quantity of the antigen or antibody is then estimated ~rom the results of the amount o~ the fixed complement components.
In the hemolysis, complement components act on the sensiti~ed erythrocytes including sheep red blood cells and anti-sheep red . "~. ' - ' - - . : ., i: ~
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blood cell antisera so -tha~ the conlplemen-t components may be de-termined while using the hemolysis of the sheep red blood cells as tha index. Ilowever, practica:L de-termination operation of the hemolysis ls 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 de-termination 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. ~3498/1980 discloses one of such 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 with an en~yme, 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 method involves -two step reactions, since a labelled antibody which binds, as an antigen, ~O a complement component must be used. Accordin~ly, rinsing operations are required after each of the reactions, leading to increase in labor and time. In -fact, this determination oethod costs much time as several hours.
On the other hand, a method of determining a . . . :, .

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7~3 neutralizing an-tibody has been made known~ -for example, by Takashi Kitamura, '~Tissue Culture Technology for Inspection of Virus", published by KINDh:t SHUPPAN ~ 0), page 246. ~hen an antibody agains-t poliovirus, for instance, is de-termined by this method, cultivated cells origina-ted from human being, a monkey or an ape are first inoculated wi-th the poliovirus. ~Mean~hile~ the poliovirus does never grow if it is inoculated into cells originated from the svurces other than human being, a monkey or an ape.) The cells inoculated with the poliovirus collapse and are deseased as the result o-f cytopathogenesis due to propaga-tion of the vir~. However, the reaction product of a neutralizing antibody and the viru~, (the in~ec-tiousness of virus being neutralized by the neutrali~ing antibody), can not propaga-te even if i-t is inoculated upon a cell originated from human being or monkey so that the cell is kept to have normal form and functionsO Making use of this principle, a specific virus is reacted with blood serum and then the titre of the neutrali~ing antibody is determined by inspecting the presence or absence, and the degree if present,of plaque and CPE (cytopathogenic effect).
However, ~hen the poliovirus is determined by the method described in the preceding paragraph, the poliovirus must be cultivated for about 7 days in a normal test in addition to the fact that the inspection and judgement o~ the , .: . ,, , : ~" ' - , , , ,:

: , , ~, . ~. : '. , ~L~ L6~3 result should be made by a skilled person rather than being easily conducted by a person having ordinary or midd]e level skill For this reason, an order of -tes-t -for the de-termina-tion o~ neutrali~ing antibody is no-t accepted even by a large scale inspection center at -the presen-t day.
On the o-ther hand, as a method ~or de-termining anti~en~ or antibodies in a simpler way, there has been known in the art a method wherein properties of complement component Clq binding an antigen-antibody complex is utilized.
(Simpson et al.,"Jounal of Immunological ~ethods", Vol. 67, 167 to 172 (1984).) In this known method, glutalaldehyde or periodic acid is conjugated to the complement component Clq as a cross-linker, and peroxidase (enzyme) is conjugated via said cross-linker to the complement componen-t Clq as a marker.
1~ ~archalonis J. J.7 "Biochemical Journal", Vol. 113, pp229 to 305 (1969) discloses a method in which radioactive iodine is conju~ated to the complement component Clq through the chloramine T method as a marker. However, in these known methods, an en7yme or radioac-tive iodine is coupled ~ith each of the complement component Clq molecules vîa an amino group present on the molecule generally and at random, resulting in entire modification of the molecule since the very site o~
each molecule having inherent properties capable o~ binding to an immunocomple~ has been chemically modi-fied by said cross~

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~L%~ L~3 linker or coupler Accordingly, the binding ac-tivi-ty of such a marker-labelled complement component Clq for binding to an antigen-antibody complex is seriously lowered -to a level no-t to adapt for quan-tita-tive measurement as a reagent. Moreover, a false-posi-tive reaction takes place frequently by -the latent presence of said cross-linker in the marker-label].ed complemen-t component Clq to make it impossible to continue the determina-tion operations. It has, thus, been impossible to provide a reliable determination me-thod for determining an antigen or antibody in a precise and reproducible manner by the use of the complement component Clq.
08JECTS AND SUMMARY OF THE INYENTION:
It is, therefore, a primary ob~ect of this invention to provide a method for measurement, using a complement component Clq which is conjugated with a variety of substances, such as markers, while preserving its inherent binding capacity for binding with an immunocomplex ~ithout any detraction.
Another object of this invention is to provide a method for measurement, using a complement component Clq which is conjugated ~ith a variety of substances for determining or detecting a specific antigen present in a body fluid or held or bound to a cell or body tissue or for determining or detecting the corresponding antibody for the specific antigen, a modified -' -` ~'. '''' ' ' .' ;:' ' '~, ~

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l~ ~t;LL fu~-ther obJect oE this invention is to provide a determinat;on methotl uti1i~ing a complement componen-t C1q, ~hich is simple and easy in determlrlat;on operal;ion an~ carl b0 corlducted fol a ~holt operation t:ime even by an unskille~ person.
Ye-t a Eurther object of -this lnven-tion .i5 to provide fl determination method ut:ilizing a complemen-t componen-t Clq, which has high sensitivity -to g:ive reproducible result of determination.
The above and o-ther obiec-ts oE -this invention will be apparent from the following detailecl description thereo~.
Provided in accordance with the invention is a method or measurement by the use of a complemen-t component Glq comprising reacting a maker-labelled complemen-t componen-t Clq with a material to be measured, said complement component Clq being coniugated with -the marker via a sulfur atom at at least one site not involved in binding immunoglobulins, thereby to obtain a reac-tion material having said marker, ancl measuring said marker.
BRIEF DESCRIPTION QF THE DRA~ING:
The sigle figure, Fig. l, of the appended drawing is a graph showing the change in neutrallzing an-tlbody titer of an anti-HSV positive human serum in Example 12, one o-f the e~amples of the invention.

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The present inven-tion will now be decribed in de-tail.
In general, after an antigen is bound to an antibody, a complement is bound -to the antibody already bound with the antigen to destruct -the an-tigen. The complement includes complement componen-ts Clq, Cls, Clr, C2a, C2b, C3a, C3b, C4b, C5b, C6, C7, C8 and C9/ and each one of these complemen-t components binds to a specific antibody pertinently depending on the specific immunological reaction or allergy rection. These somplemen-t components bind in a -fixed order such that the component Clq binds to the antigen at a first place, followed by binding of Cls and Clr to Clq, and then the other complement components bind serially.
After eager investigations with the estima-tion that a substance-conjugated complement ccmponent having a u-tility when used as a determination reagent might be prepared by conjugating a variety of substances to the complement component Clq, which is the component coupled with an antigen-antibody complex at the -first place, such that the subsequen-t binding between the immunoglobulins acting as an antibody and the component Clq is not hindered, we have succeeded to allow a varie-ty 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 ,. , :. .

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~Z7G~3 nine S-S bonds located a-t -the sites of the polypep-tide molecule of the complement componen-t Clq, -the S-S bonds loca-ting a-t the sites remo-te enough for affecting the si-te having the binding capability for the immunoglobulins and being formed by -the ourth cysteine residues from the N-terminals of the ~, B and C chains of the polypeptide constituting the complemen-t component Clq so that they are apt to be attaked by a reducing agent and apt to conjugate wi-th 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 o-f a reducing agent to expose at leat one S-H group in a first s-tep.
The reducing agents ~hich may be conveniently used in this step include those used conventionally, the examples being sulfur-containing compounds, such as `15 mercaptoethylamine, dithiothreitol, 2-mercaptoethanol~
cysteine and glutathione.
The reducing step is carried out under the condition that the complement component Clq is not modified. Preferably, reducing may be effected by dissolving the complement component Clq in a buffer solution in ~hich it exists stably and then it is attacked by a reducing agent, Examples of the buffer solution used for this purpose~lnclude a tris buf~ered saline containing lO~ of sucrose, l mol of sodium chloride and 5 mM
(millimols) of sodium ethylenediamine tetra-acetate, and a . , . ,. ~i , , . . . ~ .. . . .
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~27~13 phospha-te bu~fered saline (PBS). The reducing reaction is carried out, ge~rally, at about -2~ -to 45~C ~or about 30 seconds -to ~4 hours, the reaction tempera-tur0 and time being changed depending on the speciflc reduc:ing agent used.
I-t is desirous -that -the thus reduced complement component Clq be stored in a buffer solution -to be used in the subsequent step af-ter removing the excess reducing agen-t by means of a conventional method, such as dialysis, salting-out proc.ess or gel filtration.
The complement component Clq utilized in the present invention is a glycopro-tein contained in the blood serum o~
animal, and has nature for binding firmly to the immunoglobulins when the immunoglobulins contained similarly in the blood serum and aoting as an antibody react speci~ically ~ith 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 fraction enriched in the Cl~ component may be picked up in accordance wîth a conventional purifying operation. (In this connection, reference should be made to "Operations in Immunological Experiment B", published by the Japanese Immunological Society, pp 1376 to 13B0 ~1~74), if necessary.
In the process of the invention, a variety of substances is conjugated via the exposed SH group of the :

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~ . ' . ', ' ; ' 31 ~76~ 3 reduced complement component C14 at -the subsequent step~
The substanc.es to be used and -to be conjuga-ted with -the conlplenlent component Clq via the exposed SH group include signal emi-tting substances, such as enzyme subs-trates, dyestu-ffs, magnetizable substances, donors or accep-tors for electron transference, radioactive materîals, me-tal compounds and metal compositions, which emit signals detec-table by the sensory or~ans or ex-ternal instruments, or enzymes or coenzymes which may be modified to emit detectable signals.
More specifically, examples of the enzyme substrate are o-nitrophenyl- ~-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 wide variety of substances which take part in the electron transference, and chlorophyll which may be energized to -take ~0 part in the electron trans~erence is included in this group of substance and preferably used in the inventionO Examp].es of the radioactive substances are 1~4I-labelled albumin, p-chloro(Z~ 3 Ng)mercuribenzoic acid, N-ethyl~2,3_1~C)maleimide and iode(1-14C)acetamide.

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The me-tal compounds and composi-tions, o-ther -than -the corbonic iron referred -to hereinabove, which may be llsed ;n -the invention include gold colloid and iron-coTltaining microbeads.
Examples of -the enzymes are peroxidase, alkaline phospha-tase, galactosidase and alcohol dehydrogenase; wheras examples of the coenzymes are n.icotinamide adenine dinucleotide~
nicotinamide adenine dinucleotide phosphate7 flavin adenine dinucleotide and flavin adenine dinucleotide phosphate.
For instance, an en~yme may be utilized as the signal emit-ting substance while bein~ conjugated to the co~plement con~ponent Clq to act as a marker so that it is used as a determination or de-tection reagent for determining or detecting a specific antigen present in a body fluid or held by or adhering to a cell or body ti~sue, or for determining or detecting the corresponding antibody to said antigen, modlfied immunoglobulin or an immunocomplex.
In the aforementioned step, 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 havin~ a coupling -function, for example, via one or more spacers or cross-linkers. Examples of the substance which ~ay be directly conjugated include a ~
activated thiol Sepharose ~Trade Na~e-of Pharmacia Fine . : : .
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~27G103 Chemicals Go.) and p-chloromercuribenzoate. Any subs-tance having a group capable of conjugating -to -the exposed -SH group of the complement component Clq, such a~ maleimide residue or -SH group, and also having another group capable of coupling with the substance to be coupled indirectly ~i-th the complemen-t component Clq may be used as -the coupling agent for such purpose.
The coupling agent or -the second subs-tance having a couplin~ function, of course, varies depending on the substance to be coupled thereby. As an illustrative example, ~hen peroxidase extracted from horseradich is coupled by the N-hydroxysuccineimide ester of N-(4-carboxycyclohexyl-methyl)maleimide, both reagents are dissolved in a buffer having a pH value of 6.5 to 7.5 and con-taining sodium ethylenediamine tetra-acetate to allow them to react with eath other at 30&~ for an hour. The reaction conditions are selected in consideration of the characteristics of a cross-linker used.
Other than the N-hydroxysuccine1mide ester of N-(4-carboxycyclo-hexylmethyl)maleimide referred to above, N-hydroxysuccineimide esters of m-maleimide benzoic acid, N-(4-carboxyphenylmethyl)-~O maleimide and maleimide acetic acid may be used as the cross-linker. A peroxidase having therein a maleimide group is 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~

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~ 7~L~3 sodiunl ethylenediamine tetra-aceta-te a-t ~C -for 20 hours, whereby a complement component Clq conjuga-ted ~i-th peroxidase is obtained.
At the final step9 the fraction of the complement component Clq labelled with peroxidase, which serves as a marker~ and having -the activities originated both from the pero~idase and the complement component Clq may he picked up through the gel filtration.
It is preferred that the coupling step a-fore-mentioned be carried out in the presence of a buf-fer ~or both o-f the reduced complement component Clq and the substance -to be conjugated thereto ~ and carried out in the presence o-f a buffer ~or the coupler or cross-linker in case where such a substance having the coupling or cross-linking function is used.
In the subs-tance-conjugated complement component Clq provided accordin~ to this invention~ the substance is conjugated at a site or sites having no bindin~ capacity with the immunoglobulins, so that the bindin~ capabilty to th~
immunoglobulins inherent to the complement component Clq is 2~ kept intact without being hindered by the coniugating substance.
Since the substance-conjugated complement components Clq provided according to this invantion are conjugated ~ith various signal emitting substances without blockin~ the sites at which the immunoglobulins is to be bound, they may be used for .- ~ . ..
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~2'79~ 3 ~arious applica-tions including de-termination or measuremen-t reagents giving the reproducible results in which -their inheren-t capaci-ties for binding with the immunoglobulirls are u-tilized.
The me-thod for -the measurement or de-termina-tion, according to -the invention, u-tilizing the substance-conjugated complement component Clq will now be described. The signal emitting substances, as described hereinbefore, may be used in the measurement method according to the invention~ Such a signal emit-ting substance may be utilized as a marker~ In detail, a substance-conjugated complement component Clq having a marker conjugated at a si-te that is not adapted to bind with the immunoglobulins is prepared, and then the thus prepared marker-labelled complement component Clq is allowed to react ~ith another material to be measured, whereby a reaction 1~ product coniugated ~ith the marker is obtained. Subsequently, the labelled marker is qualitatively or quantitatiYely analysed to measure a variety of antigens, antibodies, neutrali~ing antibodies, substances produced or appearing in cells or on the surfaces of cells or microorganisms. In this manner, the method of the invention may be applied for comprehensiYe uses, including various clinical inspections and diagnoses of diseases.
One group or category o-f the substances which may be measured by the method o the invention includes - . . .. . ~

, . ' , ~27~ 3 complement-binding antibodies. By the determina-tion o~ certain complement-binding antibodies, various diseases of wide-ran~ing hosts including no-t only human beings bu-t also animals and plants may be diagnosed and judgement may be made whether the hosts are infected or not, the diseases ~hich may be determined by the method of the invention including infec-tious diseases caused by microorganisms, such as bacterium, chlamydia and virus, tumor and autoimmune diseases such as sys-temic lupus erythematodus Particularly in clinical inspection of such an infectious disease and autoimmune disease, it is a common practice to inspect ~hether or not a specific antibody uniquely appearing ~ith a cer-tain 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 important inspec-tion methods. Not~i-thstanding that this complement-binding reaction has a u-tility ~hen utilized in a method of inspecting a certain specific antibody, the reaction has not been frequently uesd, irrespective of the importance thereof, since i-t involYes cumbersome determination operations and requires extremely high level skill.
Under these circumstances, a considerable contribution in industrial and medlcal 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 ~ . - , , -. . . .

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~Z~7~i~L~33 high sensitivity.
In determina-tion o~ a complement-binding antibody in accordance ~ith the present invention, an antigen is initiall~
~i~ed to a solid phase or carrier, and -then the fixed antigen is allowed to reac-t ~ith an antibody and with a complement component Clq combined with a marker, followed by removal of unreacted materials and then the marker is quantita-tiYely analysed. Utili~able antigens include viruses and bacteria, such as varicella-zos-ter virus, measles virus, rubella virus, influenza virus, herpes simplex virus, hepati~tis 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 1~ operations, particularly rinsing operations 9 can be carried ou-t easily as compared with the prior art technology in which a lquid phase mus-t 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 microtiter plate and polystyrene beads may be referred to as illustrative ffxamples. The antigen may be fixed to the solid phase by fixin~ the same on the surface of the solid carrier - , ,, ~, . . - ......... .. .

~ 7 ~L~3 through physical absorption or chemical covalent bond and the like. In case where a cell or tissue is used as a solid carrier, the an-tigen may also be fixed -thereto by infec-tion.
Then7 -the fixed antigen is allo~ed to react wi~h the specific or corresponding antibody and a complemen-t component Clq combined with a marker. The antibody is the objective substance which is to be measured or inspected by the method of the invention, according to the aspect of the invention discussed just now, and -the exampl0s -th0reof are body fluîds, such as blood serum, cerebrospinal fluid and saliva~ The blood serum is used most frequently, since it contains the largest quanti-ties of antibodies. The antibodies determined or measured by the method of the invention must bind to the corresponding antigens uniquely or specifically, and must be capable of binding to the complement componen-ts.
However, almost all of the antibodies produced in living bodies satisfy the aforementioned conditions, and henc0 they may be measured by the method of the invention.
The reaction of the antigen fixed to the solid phase, ~O the antibody and the complement component Clq conjugated ~ith a marker takes place spontaneously by simply mixing the aforementioned three reactants toeether to complete the reaction for forming a combined product. While the reac~ion temperature and reaction time vary depending on the specific - ~., : , .- , . -,. ~,:: ': . ,. ,.':.. ' .. , - . ' ' .. .. . . . . ~
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~L~7 6~L6~3 an-tigen or other reactan-ts used, these conditions maY be selec-ted properly unless bioactivities are los-t.
Since the complex composed o-f the antigen, the antibody and -the marker-labelled complement component Clq is fi~ed to a solid phase, the unabsorbed complement component Clq and inhibitors for the reaction can be readily removed by rinsin~. The marker incorporated in the complex is then quantitatively analysed. For quantitative analysis, any of the known methods may be used, including visual measurement, observations through various types of microscopes, measurement of absorbance of visual and ultraviolet rays, fluorophotometric measurement and pulse count measurement. In determination of the marker, oth~r than the direct measurement of the marker labelled to the combined Gomplex, 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 quantitative analysis of the marker, the quantity of the complement-binding antibody coupled to a specific antigen can be learned.
In the measurement or determination method o-f the invention, en~ymes are particularly preferred as the signal emitting substance. Since enzymes act as catalysts, the sensitivities of the measurement may be freely adjusted by : : ,. .
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~7$~3 changing the temperature and time of the reac-tion.
Yarious an-tigens may also been inspected by -the measuremen-t method of -the inven-tion. The measurement of a variety of antigens may be utilized for the de-tection, identifica-tion, quantitative analysis and inspection of various substances including microorganisms such as chlamydia and virus, various physiologically active substances such as interferon and lymphokines, specific an-tigens for cancers, specific antigen substances in immunological abnormalities, allergens in allergic diseases, and m0dicinal subs-tances such as hormones; and thus the method may be applied for various purposes, such as speedy and reliable diagnosis on a Yariety of diseases, provision of the standards for judging the effects of curing actions, inspection for doping, inspection for the determination of foreign mattsrs in products~ and hygienic or sanitary inspections.
When a specific antigen is measured by -the method of the invention, a substance havin~ affinity with -the antigen which substance is fixed to a solid carrier is reacted with the ~O antigen, and the complement component Clq labelled ~ith a marker7 and optinally with an antidoby, followed by removal of unreacted materials and then the marker is quantitatively analyesd.
The substances having a-ffinity with the antigen include various types of substances by which the antigens , - . : - : : .: . . .. - . :
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~ 27~3 are readily absorbed, examples thereo-f being an-tibodies, portions of antibodies containing the sites binding to the antigens ~for example, Fab, F(ab' ) or F~ab' )z], enzyme substrates and inhibitors, protein A o~ staphylococcus3 various medicinal substances originated from organisms, and receptors for virus. ~ living tissue containing a substance having affinity with a specific antigen may be used without being purified. A selected one of these substances having affinities with antigens is fixed to the solid phase or carrier. By the use o-f the fixed phase, rinsing and other opera-tions can be simplified. Any carriers may be used for this purpose, as far as the su~stances having affinities ~ith 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 antigen is conjugated with the subs-tance. Any antigens may be measured by the method of the invention without particular ~O limitation, as fas as they can be coupled with substances having a-ffinities thereto3 such as the corresponding antibodies.
Illustative antigens which may be measured by the method of the invention include microorganisms such as viruses and bacteria, products produced by viruses and bacteria, vital :

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componen-ts in anill~al tissues, physiologically actlve substances of plan-ts~ and chemicals. Sources for such an-tigens are body fluids such as blood, ~Irine~ cerebrospinal fluid and saliva, processed products o~ meats and plan-ts, and aq~leous solutions from rivers, se~ege or waste water.
In the method o-~ measuring an antigen, according to the invention, 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 substanc0 having the af-finity with the antigen, it is not requisite to fur-ther add -the antibody.
However, in case where a susbstance having no complement-binding activity is used as the subs-tance having the affinity with the antigen, it is essential to add the antibody to be bound to the marker~labelled complement component Clq.
The antibody may be added at any desired time point after the antigen is added, and may be added simultaneously wi-th -the addition of the complemen-t component Clq or may be added before or after the addi-tion o~ the complement component Clq.
Of course~ the antibody should be the one ~hich binds uniquely to the specific antigen to be measured and should be capable of binding with -the complement component Cl~. Typical antibodies used commonly are immunoglobulins contained in animal blood sera, the examples being IgM, Ig~, etc. having : .

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complemen-t-binding activities. NaturaL antibodies present in blood sera may be used, or desired antibodies may be ob-tained by administrating or infecting animals wi-th antigens. In addi~ion to the immunoglobulins puri-fied and separated from blood sera, inactivated blood sera may be used as the an-tibodies ~ithout purification.
The condition -for the reaction bet~een the an-tigen, the substance having af-~inity with the antigen 9 and the marker-labelled complement component Clq and the antibody if it is added is not ristricted. Only by mixing the materials7 the reaction proceeds spontaneously and quantitatively. While the time and temperature of 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 biolo~ical activities of the reactants are preserved.
Since the comlex o~ the substance having a-~inity ~ith the antigen, the antigen and the marker-labelled complemen-t component Clq is fixed to the solid phase, the unreacted complement component Clq and inhibitors for the reaction may be easily removed by simple rinsing opera-tion. The marker o~ the complex -fixed to the solid phase is then quantitatively analysed.
For the quantitative analysis, similar methods as has been described in determination o-f complement-binding an-tibody may be employed.

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~:7~ 3 Neutralizing antibodies may also be measured by the method of -the invention. Neu-trali~ing antibodies are antibodies for preventing in-fections by microorganisms, such as virus, rickettsia and chlamydia. Re-ferring to diseases caused by virus, for instance, a ~ide variety of viral infectious diseases have been known up to date, including not a few sarious diseases. For instance, if a pregnant woman is infected with rubella virus, there arises a danger that a mal~ormed baby is born. Fatal damages are caused by the infection with rabies virus, Japanese encephalitis virus and poliovirus, with the nerval cells suffered unrecoverable disorders, leading to lasting troubles throughout the lifetime.
Hepatitis caused by hepatitis virus is an infectious disease 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.
However, 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 counter-measure against the diseases caused by virus to prevent infection by virus or to protect a person rom infection, apart from the curing treatement of the patients. The judgement on the " ` ' `'` '. -', '' `'' . .

~ 7 ~L~3 question whether a person is susceptible -to infec-tion by a speci~ic virus or no-t may be rendered by -the de-termina-tion of presence or absence of -the neu-tralizing antibody to the virus under question and by the measur~ment o~ -the ti-ter of the existing neutralizing antibody.
A living body acquires sound immunity after it has been infected wi-th a specific virus and then recovered from the disease caused thereby. This means, in facty that a system for preventing the living body from re-infection with that virus has been established. In other words, a living body has been once infected with a specific virus, the antibody for protecting the body from re-infection is promoted, the antibody being referred to as infection preventing antibody.
Production and preservation of the infection preventing ~5 antibody are very important factors against the infection by the virus. After being infected or immunized ~ith a virus against the atta~k by the virus (for example inoculated by vaccine), various antibodies against the structural components of the virus have been produced in a livin~ body. However, all of these antibodles produced in the living body are not participated in the prevention against infection by that ~irus.
Only the antibody having the f~nction for inhibiting the gro~th or propagation of the virus is referred to as neutralizing antibody or infection preventing antibody. This particular ~, ~ ' ` ~ ~. ................................ .

, . . . . .

neutralizing an-tibody e~erts the principal role in preven-tion of infection. The present invention provides a me-thod o~
measuring a varie-ty o-E neu-tralizing antibodies agains-t all viruses, rickettsias and chlamydias which in~ect cul-ture cells speedily and quanti-tatively on a number of samples.
In the method of measuring a neutralizing antibody, according to the invention, a liquid con-taining a known quantity of microorganism, such as virus, rickettsia or chlamydia, is reacted with a body fluid to be measured, such as blood, cerebrospinal fluid, saliva or blood serum. At this reaction step, the neutralizing antiboy, if present, reacts ~ith the mixed microor~anism . The amount of microorganism reacting with the neutralizing antibody is increased as the amount of neutrali~ing antibody contained in 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 amount of neutralizing antibody contained in the measured liquid can be calculated from the result of determination of the residual microorganism.
After the precedin~ reac-tion step, the residual microorganism is inoculated on culture host cells to allow to grow. The:cultivation is stopped after the lapse of ~re-set time, whereby fixed cells containing therein the residual microorganism are obtained. A marker-labelled complement - 25 - :

.

,, , ~ , :
.
.

~ 7~ L~3 component Clq and an an-tibody agains-t the microorganism are then added to react wi-th -the fixed cell -to ob-tain a modified ~ixed cell to which -the marker-labelLed complement component Clq and the an-tibody are bound By the quan-titative analysis of -the marker, -the amount of residual microorganism can ~e learned to find the amount or titer of the neutralizing antibody.
The residual microorganism is, in general, cultivated initially by inoculating the microorganism on host cells cultivated through a monolayer culture on a micro plate to allow the microorganism to be absorbed 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 trea-ting with, for example, methanol-containing hydrogen peroxide~ Antibodies a~ainst the residual microorganism which may be used in the method include antisera, such as low titer human sera and animal immunoe sera, and monoclonal antibody.
Furthermore7 the materials which may be measured by the method of the invention, other than those described above, are products produced or appearing internally o or on the surfaces of cells,and various microorganisms. The materials belonging to this category include cell surface anti~ens produced by cells7 such as asialo GMl, T antigen and Ly antigen;
in-tracellular en~ymes, such as TdT (-terminal deoxynucleotidyl - 2~ -. . .
- . . : ~ , .

.

~.27Ç~

transferase) 7 GTP (V -glutamyl -transpeptidase) and L~H (lactate dehydrogenase); secre-ting substances, such as CEA (carcino e~bryonic antigen) and A~P ~ -fetopro-tein) and immunoglobulins;
and enzymes and pep-tide base substances produced by yeasts and bacteria. The cells referred to above include all kinds of cells including animal cells, plan-t cells, heterokaryotes, cells of yeasts, bacteria and protozoa, and cells subjected to gene engineering. Microorganisms ~hich may be measured by the metl1od of the invention include all microorganisms infecting cultivated cells, such as viruses, rickettsias and chlamydias.
In measurement, one of the aforemen-tioned substances or microorganisms is cultivated and fixed, or simply fixed, and then reacted with a complement-binding antibody and a marker-labelled complement component Clq, followed by determination of the marker9 whereby the substance or microorganism may be quantitatively analys d.
EXAMPLES OF THE INVENTION:
The invention will now be illustratively ~escribed with reference to examples thereof and comparative examples.
~o Experiment Enzyme-Conjugated Complement Component Clq:
(l) Purification;
lOO m~ o-f a fresh rabbit blood serum was diali~ed through 5 Q of a 0.026M aqueous solution of ethylene glycol -::: : . , : ' ' ` : ' '~ , - ,. . .
: . ; : . , -~276~3 tetra-acetate (p~l 7.53 -for 15 -to 24 hours, and -the formed precipitate was recovered by centrifugal separa-tion ('~O,OOOG, 20 minutes)O The recovered precipitate was dissolved in 20 m Q
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 cen-trifugal separation ~25,000~, 30 minutes), and then the solution was diali~ed through 5Q of 0.063M aqueous solution of sodium ethylenediamine tetra-acetate (pH 5.0) at 5~ for 4 hours, followed by removal of precipitatas by centrifugal separation ~20,000G, 20 minutes). About 3 mg of proteins were obtained by the aforementioned operations~ and 95~ or more of the thus obtained proteins was occupied by the complemen-t component Clq.
In order to store the complement component Clq, the proteins were dissolved in an aqueous solution ~pH 7.4) containing 0.05M tris~hydroxymethyl~aminomethane, lM sodium chloride, 0.005M sodium ethylenediamine tetra-acetate and 10X sucrose.
The aforementioned operation se~uence may be repeated to further purify the complement componen-t Clq.
~2) Preparation;
30 mg of the -thus purified rabbit complement component Clq was dissolved in 10 m B of an aqueous solution ~pH 7.4) containing 0.05Y tris(hydroxymetbyl)aminomethane, lM sodium chloride, 0.005M sodium ethylenediamine tetra-acetate and 10 .

. : . . ~ - . ;. .

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

~ 2~6~

sucrose. The solution was then added with o.l ml of a o.lM
dithiothreitol, and allowed to stand a-t room temperature for an hour for reaction. The reaction solution was then passed through a Sephadex G-2s (Trademark of Pharmacia Fine Chemicals Co.) column to recover the protein fraction which was concentrated to have a volume of about 10 ml by ultrafiltration to obtain 2~ mg of a reduced complement component Clq.

Separately, 20 mg of peroxidase extracted from the horseradish ~as dissolved in 6 ml of a phosphate buffer (pH 7.~), and then added with 4 ml of dimethylformamide. The solution was further added with 0.2 ml of a 2% 4-(maleimidemethyl)-cyclohexane-l-carboxylic acid succuneimide ester (hereinafter referred to as CHM) in dimethylformamide, and then allowed to stand at room temperature for an hour for reaction. After an hour, the solution containing the reaction product was passed through a Sephadex G-25 column to recover 16 mg of a CHM-con~ugated 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 at 4C to 10C for 15 hours and then passed through a Sepharose 6B (Trademark 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.

~5 Experiment 2 Enzyme-coniuqated ComPlement ÇomPonent Cl~:

1 mg of ~-D-galactosidase derived from Escherlchia coli was dissolved in 0.2 ml of a O.lM phosphate buffer ~pH 6.0), and then reacted with Ool mg of N,N'-o-phenylenedimaleimlde dissolved ln 0.2 ml of a phosphate buffer containing 5% dimethylformamide at 30C for 25 minutes. The solution containing the reaction product ~c .. . .

~L~7~ 3 was passed -through a Sephadex G-2s ~Trademark of Pharmacla Fine Chemicals Co.) column equilibrated wlth a phosphate buffer containing o.2 mg/ml of bovine serum albumins, whereby a 720 ~g of ~ -D-galactosidase coupled with maleimide.

200 ,~g of the ~ -D-galactosidase coupled with maleimide was dissolved in 0.1 ml of a phosphate buffer containing 1 mM of sodium ethylenediamine tetra-acetate, and reacted with 2 mg of the reduced complement component Clq prepared in Experiment 1 and dissolved in 0.1 ml of a phosphate buffer containing 1 mM of sodium ethylenediamine tetra-acetate at 4C for 48 hours. The solution containing the reaction product was sub~ected to gel ~iltration using a Sepharose 6B (Trademark of Pharmacia Fine Chemicals Co.) column, and then processed through the procedures as described in Experiment 1 to obtain an active fraction, i.e. a fraction containing 1.6 mg .
: : :' . . .
:. - .. ~. : . .
. : . . ..

, ~27~3 of a complement component lq combined with ~-D galac-tosidase.
Example Enzyme-conjugated Complement Componen-t Clq:
~1) PrePara-tion;
Generally following to the same procedures as in Experiment l~ except that a goat serum was used in place of the rabbit serum, a reduced complement component Clq ~as 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 16, 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 o-f this Example ar0 shown together with -the results of the following Comparative Example 1.
Comparative Example Enzyme-conjugated Complement Component Clq Prepared by Conventional Process and Having the Enzyme Conjugated Generally at Random:
~l) Preparation;
1.5 mg of horseradish peroxidase was dissolved in ~-' ' '' . " ". ' ,':' ' ;
:
. . . . . . .. : .
- ., ~. ~, . . .
, ~ -, . . ~,', ' ' , . : -: ' : . :

76~L~3 0.~ m Q o:t` distilled wa-ter, and added with 60 ~ Q of a O.lM
sodium periodate solution9 -followed by agitation at room temperature for 20 minutes. The solu-tion was dialized through an acetate buffer (pH ~.4) con-taining lM sodium chloride, added with 60 mg of sucrose, and -then added with 1 m Q o-f 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 was further added with ~.1 mQ of a 4 mg/mQ solution of sodium borohydride, and then allowed to stand at 4C for additional 2 hours.
Thereaf-ter, the admixture was subjected to gel filtration through a Sephacryl S-30~ ~ of Pharmacia Fine Chemicals Co.) column, and the fraction having both of the peroxidase activity and the Clq activity was collectedO
(2) Test;
Usin~ a serum having a CF antibody titer of 16, 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 en~yme 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 o-f Example 1.

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

. ~
- - , : , :

~27~

Table Color Developmen-t Color Development o-f Herpes CF of Normal Antigen Well Antigen Well _ Enzyme-Labelled Clq 0.544 0.078 of Example 1 En~yme-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 with the herpes simplex virus CF
anti~en selectively or uniquely; whereas the enzyme-labelled complement component Clq prepared by the process of the invention is considerably lowered in inselective or non~unique reaction ~ith the normal antigen to have a sufficiently high capability of reaction ~ith the herpes simplex virus CF antigen.
Example 2 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).

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

7~3 ~fter reacting the mixture for 7 hours, -the reaction mixture ~as subjected -to gel filtra-tion to obtain bovine serum albumin combined with FITC. ~.2 mg of -the bovine serum albumin combined with FITC was then dissolved in 0.4 m Q of a O.lM
sodium phosphate buffer (pH 7.0), and added with 50 ~ Q of a 90 mg/mQ solution of C~IM in dime-thylformamide for reac-tion at 30&~ for an hour. After removing the insoluble materials by centrifugal separation, the buffer solution was exchanged to a O.lM phosphate buffer (yH 6.0). 0.56 mQ of the solution `lO was added ~ith 0.5 m~ of a O.lM phosphate buffer (~H 6.0) containin~ 8 mg of the reduced complement component Clq prepared in Experiment 1 and 5 mM sodium ethylenediamine tetra-acetate, and the admixture was maintained at 4C for 18 hours. After then~ -the admixture ~as filtered through a gel filter of Sepharose 6B column to obtain 12 mQ of an eluate fraction having a molecular weight ranging within 400,000 to 900,0~0 and containing a reaction product between ths complement componen-t Clq and the bovine serum albumin combined with the FITC.
~2) Test ~O Separately, the spleen cells ~ere removed ~rom the B~F1 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 ~4 ..

- . - , .

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

~L2 7 Ç~9L~3~3 mixed toge-ther -to form a mi~tu-re having a concentra-tion of 150 times of -the -final concen-tra-tionO After maintaining the mixture on ice fo~ an hour and rinsing sufficientlyt -the cell was floa-ted on the surface of a 50~ glycerîn-phosphate buffer solution and observed -through a fluorescen-t microscope. The result was that ~3X of the cells emi-tted fluorescent light -to reveal that almost all of the cells ~ere the T-cells.
Example 3 Complement-Component Clq Conjugated with Donor or hcceptor for Electron:
(1) Preparation;
Chlorophyllin a was dissolved in distilled wa-ter so that a 1 mg/mQ solution was formed, and the pH value of the solution was adjusted with hydrochloric acid to pH 7.5, followed 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 0.8M of the latter.
The mixture ~as allowed to stand for reaction for 120 minutes.

The reaction product was purified by the use of CM-Sephadex ~ ~ ~4Q ma~
2Q (ir~k~hr e of Pharmacia Fine Chemicals Co.), and 450 ~ ~ of aminoethylated chlorophyllin a ~as dissolved in 0.5 mQ of a O.lM phosphate buffer (pH 7.0) containing 40% of dimethyl-formamide~ The solution of purified aminoethyla-ted chlorophyllin a was mixed with 0.5 mQ of a 40X dimethyl-, , - . : . ` ' ' ~2~ 3 formamide solution con-taining 2% of CHM, and maintained at 30C for an hour to react wi-th CIIM~ The reaction mixture was ~-~ subjected to gel filtra-tion using Biogel P-2 (-~x~h~ ~ of Bio-Rad Laboratories Inc.) to ob-tain CtlM-chrolophyllin a.
12 mQ of the reduced complement component Clq prepared in accordance with the process as described in Example 1 and 100 ~ g of the GHM-chrolophyllin _ were put into 1.5 mQ of a OolM
phosphate buffer ~pH 6.0), and allowed to stand stationarily at 4~ for 18 hous, follo~ed by gel filtration at which a fraction having a molecular ~eight ranging within 40Q,OOO to 600,000 ~as picked up, whereby 7.5 mg of chrolophyllin-labelled complement component Clq was obtained.
(2) Test;
Separately, a chemically modified antigen electrode was prepared by coa-ting an antigen protein against herpes simplex virus on an SnOz Nesa electrode so -tha-t the antigen protein was combined with the electrode through a covalent bond.
The electrode was immersed in a 25 ~M phosphate buffer (pH 6.95) 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 complement component Clq, and irradiated by a whi-te light while maintaining the electrode potential at 0.1 Vs- SC~, whereupon generation of photocurrents was observed. The quantum efficiencies of - . ~

- ' - .- '- " ' ~27çi~Lq~3 photocurren-ts were about 9X.
Example Magneti~able Substance-Conjugated Complement Component Clq:
~1~ Preparation;
Polystyrene microbeads each having amino groups at the surface thereof and containing m.icro grains of magnetite were suspended in 1 mQ of O.lM phosphate buffer ~pH 7.0) containing 20~ dimethylformamide7 and added with 40 ~ ~ of 0.1~ phosphate buffer containing 2.5X CHM, followed by modera-te agitation at 30C 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 vith 0.5 m~ of another phosphate buffer containing 1.8 mg of -the reduced complement component Clq prepared by the process as described in Example 1, and then the admixture was allowed to react at 4C for 18 hours under moderate agitation. The beads were rinsed with a Veronal ~uffer solution ~pH 7.4) containin~ 0.1X of gelatin, the solution being referred to as GVB hereinafter, and then stored in the GYB at 4~.
(2) Test;
Separately, the spleen cells of X5563 tumor-bearing C3H/He mouse were cu1tivated on a culture medium containing IL-2, and the once rinsed cells were again floated on the same culture medium and mi~ed with the beads-conjugated complement - ~
- . : .
- - . . - .

- - : . ~

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

~ ~6~3 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-se-t time, the cells were collec-ted and then collected cells were again floated gently, and an intense magnetical force was applied from the exterior 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 medium 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 5 Measurement of Complement-sindinq Antibody:

On a 96 well microtiter plate absorbing a complement fixed antigen of the herpes simplex virus added, respectively, were 5, 1 of each of sample sera inactivated to have complement-binding titters of less than 4 and 16, and then 95 ~1 of the peroxidase-labelled complement component Clq in Example I (diluted to 100 times volume with a gelatine-Veronal (a Trademark) buffer solution) was added, followed by stationary standing at room ~0 temperature for an hour. Thereafter, each well was rinsed three times with a phosphate buffer containing 0.05% of Tween-20 (a ~rademark for a surface active agent produced and sold by Nakarai Chemicals LTD.), and added with 100 ~1 of an H202-ABTS solution [2,2'-- 38 - .

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

~LZ~76~L~)3 adino-di-(3-ethyl-benzo-thiazo].ine sulfate) solution containing hydrogen peroxide], followed by s-tanding at room -temperature for an hour -to complete the reaction. Af-ter adding with 100 ~ Q of an enzymatic reaction terminating agent, -the ligh-t absorbance at 414 nm was measured to find that the absorbance of -the serum having the complement-binding titer of less than 4 was 0 029 while that of the serum having the complement-binding titer of 16 was 0.579.
Example 6 Measurement of ~omplement-binding Antibody:
~ ovine blood serum albumin was dissolved in a saline solution buffered by phosphate to prepare a solution having a concentration of 20 ~ g/mQ , which was poured in each well of a 96 well microtiter plate and then maintained 1~ at room temperature for 2 hours to be absorbed by each well.
After removing free bovine blood serum albumin, 50 ~ Q o-f anti-bovine serum albumin rabbit antiserum (stepwisely diluted by 800 to ~400 times with a gelatin-Veronal buffer) and 50 ~ Q
of the ~-D-galactosidase -labelled complement component Clq synthesized in ~xperiment 2 were added, and the microtiter pla-te was allowed to stand at room temperature -for 30 minutes. After rinsing each well, 100 ~ ~ 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 tempere-ture for 60 - 3g -- : . . . : .
- . : . ~. : ~ , .
.
- . ' . '. -: . ~ . . .
.

~ 7 ~L~33 minutes~ and then 0.1 mQ of a O.lM sodium carbona-te solu-tion to terminate or cease -the enzyma-tic reaction. The light absorbances of respec-t:ive wells were measured at a wavelength of 420 nm to find that -the light absorbances were gradually varied from 0.421 -to 0.063 depending on the change in dosed amounts of antiserum.
Example 7 Measurement o~ 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 titers of 16 and less than 4) in a gelatine-Veronal buffer and with 0.1 mQ of a solu~ion of the peroxidase-labelled complement component Clq prepared in Experiment 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 mQ of an ~o o-phenylenediamine solution to react at room temperature for 45 minutes. The reaction was terminated by the addition of 2 m Q of 1 N hydrochloric acid, and the light absorbances of the samples at the waveleneth of 49~ nm were measured. The sample having the complement-binding titer of less than 4 .
~ ', " , ' " , ' ', ', . ' '. ' ' ~ , ' ' ' - ' ' , ., .' , , ' , :, , ' ' ' ' ~ , ~2~7~ 3 had a ligh-t absorbance of 0.018, whereas the sample having the complement-binding titer o-f 16 had a light absorbance of Q,~08.
Example 8 Measurement of Antigen:
A 96 well microtiter pla-te absorbing guinea pig an-ti-herpes simplex virus antibody ~Fab) was supplied with 0.1 mQ of uterus cervix s~abs of a patient, and stationarily held at room temperature for 60 minutes. After rinsing the plate ~i-th the PBS (a 0.85X saline-containing phosphate buffer having a pH ~!alue of 7.4~ for three times, each ~ell ~as added with 0.05m Q of either one of the inactivated ~uinea pig anti-herpes simplex virus sera (having -the complement-binding an-tibody titers of 16 to 32) and also with 0.05m Q of the peroxidase-1~ labelled complement component Clq prepared in Experiment 1. Theplate was held stationarily at room temperature for 60 minutes.
Then, each ~ell ~as rinsed with PBS containing 0.05~ of Tween-20 (a surface active a~ent produced and sold under such Trade ~A~ /n4r`~
rom Nakarai Chemicals LTD.), and ad~ed with 0.01 mQ of ~0 H20~-ABTS [2,2'-adino-di(3-ethyl-benzothiazolin sulfa-te~
containing hydrogen peroxide] solution to be held at room temperature for an hour for reaction. Thereafter, 0.05 mQ of a 0.05X aqueous sodium nitride which ac-ted to terminate the enzymatic reaction, and then the light absorbances of respective '`

. ' .
,:
'. . . ' ~ ' ~27~)3 sample wells a-t a waveleng-th of ~1~ nm were measured to find that the sample well -fill~d with u-terus cervix s~abs of a patient ~ho was nega-tive agains-t the herpes simplex virus ha~ an absorbance of 0.030 and -tha-t -the sample wells filled with uterus cervi~ swabs of a patient who were positive against the herpes simple~ virus had absorbances of 0 113, 0.300, 0O550 and so on.
E~a~lple 9 Measurement of Antigen:
According to a conven-tional process, a lymphocyte fraction ~as prepared from the mouse spleen cell, followed by rinsing with the PBS, and then -the concentration of the cell was adjusted to 1 X 107/mQ . 0~1 mQ of the thus prepared lymphocyte fraction, 0~05 mQ of antimouse Thy-1,3 alloserum, and 0.05 m ~ of the FITC-labelled complement component Clq of Example 2 were mixed together and allowed -to stand at room temperature for an hour. The cel]s were then rinsed thsroughly with the PBS and observed through a fluorescent microscope.
The result revealed that 37X of the cells were fluorescent.
Example 10 Measurement of Anti~en:
The lymphocytes in a blood of a leukemia patient were suspended in 1 m Q of a phosphate buffer to prepare a suspension containing 1 X 10~/m Q of lymphocytes, and the suspension was processed by an ultra-sonicator for . . . ~ . . : . .

:

- '. ' ~ ` , ' '' .

~;~7~3 2 minutes. Th~ homogenate ~as lhen clarified by centrifugal separation, and the supernatant was added ~i-th 0.5m~ of D~A
Sepharose to react -therewith a-t 37~C -for 60 minutes. The DNA
Sepharose was bound ~i-th DNA rela-ted enzymes, such as DNA
polymerase and terminal deoxynucleo-tidyl transferase (TdT).
Then, 0.1 M Q of the peroxidase-labelled complement component Clq and an inactivated rabbit anti-TdT serum7 folloved by reaction a-t 37C for 30 minutes. Af-ter rinsing thoroughly with PBS, the DNA Sepharose ~as recovered, to which 1 mQ of a solution of H202-ABTS, was added, and the admixture was reacted at 37~ for 60 minutes. Then, 1 mQ of a 0.05~
aqueous solution of sodium nitride acting as a terminator for the reaction, and the ligh-t absorbance of the supernatant was measured at a wavelen~th of 414 nm. It could be iudged that the sample having a light absorbance value of not more than 0.075 showed that the patient was negative to TdT and that the sample having a light absorbance value of not less than 0.100 showed that the patient was positive to TdT and suffered from acute leukemia.
Example 11 Measurement of Neutralizing Antibody:
Two sample sera havin~, respectively, neutralizing antibody titers of 32 and 128 to the HSV ~Herpes Simplex Virus~
were diluted with a phosphate buffer to have the volumes four . ., , .

- - . . .
:: .' ~

~7~3 times as large as the ini-tial volumes, hea-ted at 56~C for 30 minut~s to be inactivated, and then fur-ther dilu-ted wi-th -the same buffer -to have egih-t times volumes. 0.1 mQ :eor each of the thus inactiva-ted and dilu-ted sample sera ~as mixed wi-th 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 momolayer culture on a microplate, onto ~hich a mixture of ths serum and the HSV ~as added at a content of 50 ~ Q /well, and then the virus was absorbed by holding the plate in a culture filled with 0.5X 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 methanol containing 3~ of hydrogen peroxide.
After fixing by the methanol containing 3X of hydrogen peroxide, as described in the preceding paragraph, 50 ~ Q
for each of human sera having complement-fixing titers against the HSV diluted by 50 times with a gelatine-Veronal buffer (pH 7.4), respectively, of 16 and less than 4 was poured into individual wells, and then each ~ell was added with 0.~ ~ g/50 ~ Q /well of the peroxidase-labelled goat complement component Clq prepared in Example 1 and dissolved in -the same buf-fer. After allowing to stand the microplate at room temperature for ~ hours, each well was rinsed with a phosphate ~, ',' ' ., .' ',' , , :' ~ ~ ,, ' . . , . . .
.
, ~ L~3 buffer solution containing 0.05~ of Tween 20 for -three times, and then added with 0.7 m ~ /well of a HzO~-ABTS solution to develop coloring of each well which was subiected to ligh-t absorbance de~ermination conduc-ted at a ~aveleng-th of 914 nm.
The results are shown in Table 2.

Table 2 Serum for Detection OD414 of Residing Virus (1) - (2) Serum for Well (1) Well (2) Added Determination of Added withwith Normal Presnece or Absence o-f Guinea PigGuinea Pig Neutralizing Antibody Anti-HSV SerunSerum Control (Well Not Added 0.618 0.095 0.523 with Sample Serum) ~ ~................. ............................. ............................. ..................
Sample Serum Having Neutra- 0.504 0.092 0.412 lizing Titer of 32 Sample Serum:Having Neutra- 0.117 0.101 0.016 li~ing Titer of 128 _ _ E~ample 12 ~0 Measurement of Neutralizing Antibody:
An anti-HSV positive human blood serum having a neutrali3ing antibody titer of 128 and a n0gative human blood serum were diluted by four~times with a phosphate buffer, and inactivated, and then a serial dilution series diluted by ~5 4 to 512 times was prepared each for the both sera on a ., . ` . ' .
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.

~27~

microtiter pla-te provided ~ith a number of wells each having a volume of 0.1 m Q /~ell. Each ~ell was filled with O.lm Q
of a buffer containing 4 X 1Oa pfu/m Q o-f HSV. The follo~7ing procedures were the same as in Example 11 to measure or determine the OD4l~. The results are plotted in the graph illustrated in Fig.1 ~herein the abscissa indicates -the dilution rate of each serum and the ordina-te indicate the QD41~. As shown, for the positive serum, the dilution rate giving the value as large as 1/2 of the maximum OD
corresponds to the neutralizing antibody titer of 128.
Example 13 Measurement of Intracellular Substance or Microorganism:
A specimen to be inspected was picked up from a defected portions of HSV infected patient tPendedum or labia), and suspended in 1 mQ of culture medium solution containing an antibiotic. 0.1 mQ of the suspension was inoculated to t~o ~ells of a microplate in which Vero cells had been preliminarily cultivated, and further cultivated at 37~ for 22 hours.
After the completion of 22 hour cultivation, the cultivated cells ~ere fixed wi-th 3% hydrogen peroxide-methanol, and 50 ~ Q of a human serum diluted by 25 times with a gelatine-Veronal buffer ~pH 7.4),the serum havin~ an anti-HSV
complement binding titer (CF~titer) of 32 or less than 4, ~as put into individual ~ells together ~ith 50 ~ Q of a solution ~ 2~ 3 in the same bu-ffer containing 90 ng o-f -the peroxidase-Labelled complement component Clq prepared in Example 1. hfter reac-ting at room temperature for 2 hours, each well was rinsed with a phospha-te buffer containing 0.05~ of Tween 20 ~or -three times, added with O.lmQ /well o~ a hydrogen peroxide-ABTS solu-tion (pH 4) follo~ed by standing -for an hour to develop coloring, and then the reaction was terminated by the addition of 0.1 mQ of a 0.01~ sodium a~ide. 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 ~ith -the serum having a CF titer of less than 4 had a light absorbance of 0.089, From those result, it could be confirmed that the HSV
virion was present in the specimen inspectsd.
E~ample 14 Measurement of Intracellular Substance or Microorganism:
With the aim to cloning a cell producing carcino embryonic antigen~ the cell T3M-4 producing CEh from pancreas tumor was diluted to the limit (i.e. -to 1 cell/well), and then cultivated on a 96 well microplate for 16 days. After removing the culture medium solution, 0.l mQ of tripsin-sodium ethylenediamine tetra-acetate was put into each well tc float the cells, and then two plates preliminarily filled with 0.2 mQ
/~ell of a fresh culture medium solution were replicated so that ,. ;. ' . ' ' : ' .
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' ~2t76~ 3 repricas con-taining 20~ Q /well of floating cells were prepared.
The culture me~ium solu-tion in one o~ the repricas was -thrown away~ followed by fixation of -the cel]s with 3~H20z-me-thanol, and then added wi-th O.l In~ of a l/400 time diluted rabbit anti-CEA antiserum diluted with GVB (gela-tine-Veronal buffer) and llO
ng/Q.lmQ /well of the peroxidase-labelled complemen-t component Clq. After reacting at room te~perature for 2 hours and rinsing, coloring of the well was developed by the addition of a solution of the substrate of ABTS Cdiammonium ~2,2~azi)-di~3-ethylbenzo-thiazolin sulfonic acid)~. The OD~l~ of respective wells rangedwithin Q.l27 to 0.3860 The cell in the well showing the maximum OD4l4 was picked up from the other reprica, and subjected to expansion .
Example l5 Measurement of Intracellular Substance or Microorganism:
lOO ~ g ~O.l m Q ) of purified a -fetoprotein and O,l mQ of Freund complete adjuvant 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 ma-terials, ~0 lOQ ~ g (0.3 mQ ) of of AFP ~ -fetoprotein) was additionally dosed, and after 3 days of the dosage of the hFP, the renal cells were picked up and fused with NS-l cells. The cells were spread over a 96 well plate at a eoncentration or distribution density of l X 10~/mQ , From the first day to - 4,8 -': , ' ' ' ., :

.:

~27~i~C)3 -the six-teen-th day a~ter fusing, selection by the }IAT medium (hypoxantine--thymidine-aminopterine medium) was conduc-ted, and the antibody activity of the supernatan-t o-t each cul-tivated well was inspected on -the seventeenth day -to reveal that prodcu-tion of antibody was recognized at a rate of 64/948 wells and prodcution of anti-AFP antibody was recognized in -two wells.
The cells in respective wells were utilized as coa-ted specimens while being processed by 3~Hz 2 -methanol -to be fixed, and then added with 20~ Q of a goat anti-mouse IgG (~ chain selectivity) diluted by 200 times and also with 18 ng/20~ Q of the pero~idase-labelled complement component Clq, followed by stational standing for 2 hours, After rinsing thoroughly, development of coloring of each cell was effected in a diaminoben7idine solution, and the number of cells producing th~ IgG was coun$ed to find that the ratio of positive cells were 62~ and 91~, respectively.

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Claims (28)

1. A method for measurement by the use of a complement component Clq comprising reacting a maker-labelled complement component Clq with a material to be measured, said complement component Clq being conjugated with the marker via a sulfur atom at at least one site not involved in binding immunoglobulins, thereby to obtain a reaction material having said marker, and measuring said marker.

2. The method for measurement according to claim 1, wherein said material to be measured is a complement-binding antibody, and wherein said complement component Clq and said complement binding antibody are allowed to react with an antigen fixed to a solid carrier, followed by removal of unreacted materials, and then said marker is quantitatively analysed.

3. The method for measurement according to claim 2, wherein said antigen is selected from the group consisting of viruses, bacteria, physiologically active substances, and antigens against autoantibodies.

4. The method for measurement according to claim 2, wherein said antigen is selected from the group consisting of varicilla-zoster virus, measles virus, rubella virus, influenza virus, herpes simplex virus, hepatitis virus, mumps virus, micoplasma phneumonitis, interferon and an antigen to the aotuantibody.

5. The method for measurement according to claim 2, wherein said solid carrier is selected from the group consisting of synthetic high polymers, natural high polymers, cells and tissues.

6. The method for measurement according to claim 2, wherein said complement-binding antibody is selected from the group consisting of blood serum, cerebrospinal fluid and saliva.

7. The method for measurement according to claim 1, wherein said material to be measured is an antigen, and wherein said complement component Clq and said antigen are allowed to react with a substance having affinity with said antigen, said substance being fixed to a solid carrier, followed by removal of unreacted materials, and then said marker is quantitatively analysed.

8. The method for measurement according to claim 7, wherein said substance having affinity with said antigen is selected from the group consisting of an antibody, a portion of the antibody containing an antigen-binding site, enzyme substrates, inhibitors, protein A contained in staphylococcus, medical substances originated from organisms and receptors for virus.

9. The method for measurement according to claim 7, wherein said solid carrier material is selected from the group consisting of synthetic high polymers, natural high polymers, cells and tissues.

10. The method for measurement according to claim 7, wherein said antigen is selected from the group consisting of virus, bacteria, products by virus and bacteria, vital components in animal tissues, physiologically active substances of plants, and chemicals.

11. The method for measurement according to claim 7, wherein an antibody is added to said antigen and said complement component Clq for reaction.

12. The method for measurement according to claim 11, wherein said antibody is selected from the group consisting of natural antibodies present in blood sera, antibodies prepared by inoculating animals with antigens, immunoglobulins separated from blood sera followed by refinement, and inactivated blood sera.

13. The method for measurement according to claim 1 wherein said material to be measured is a neutralizing antibody, said method comprising the steps of reacting a known liquid containing a known quantity of a microorganism with a body fluid containing said neutralizing antibody, allowing to grow the residual microorganism for a predetermined period, stopping growth of said microorganism to obtain first fixed cells fixed with said residual microorganism, adding said marker-labelled complement component Clq and an antibody to said microorganism to react with said first fixed cells thereby to form second fixed cells having the microorganisms bound with said marker-labelled complement component Clq and said antibody, and then quantitatively analysing said marker.

14. The method for measurement according to claim 13, wherein said antibody is selected from the group consisting of antisera, immunoe sera of animals and monoclonal antibodies.

15. The method for measurement according to claim 1 wherein said material to be measured is the one selected from the group consisting of substances produced internally of and at the surfaces of cells and microorganisms, said method comprising the steps of fixing said material to be measured, reacting the thus fixed material with said marker-labelled complement component Clq, and then measuring said marker.

16, The method for measurement according to claim 15 wherein said material to be measured is cultivated before it is fixed.

17. The method for measurement according to claim 15, wherein said material to be measured is selected from the group consisting of cell surface antigens, intracellular enzymes, secreting substances, and enzymes and peptide base substances produced by yeasts and bacteria.

18. The method for measurement according to claim 15, wherein said material to be measured is selected from the group consisting of asialo Gm1, T antigen, Ly antigen, TdT (terminal deoxynucleotidyl transferase), GTP (.gamma.-glutamyl transferase), LDT (lactate dehydrogenase), CEA (carcino embryonic entigen), AFP (.alpha.-fetoprotein) and immunoglobulins.

19. The method for measurement according to claim 1, wherein said marker is a signal emitting substance.

20. The method for measurement according to claim 19, 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.

21. The method for measurement according to claim 20, wherein said enzyme is selected from the group consisting of peroxidases, alkaline phosphotases, galactosidases and alcohol dehydrogenases.

22. The method for measurement according to claim 20, 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.

23. The method for measurement according to claim 20, wherein said enzyme substrate is selected from the group consisting of o-nitrophenyl-.beta.-D-galactopyranoside and 3-hydroxysteroid.

24. The method for measurement according to claim 20, wherein said dyestuff is selected from the group consisting of methylene blue and fluorescein isothiocyanate.

25. The method for measurement according to claim 20, wherein said magnetizable susbstance is selected from the group consisting of carbonic iron, iron-containing microcapsules and complexes of iron with proteins.

26. The method for measurement according to claim 20, wherein said donor and acceptor for electron transference are chlorophyl.

27. The method for measurement according to claim 20, wherein said radioactive substance is selected from the group consisting of 124?-labelled albumin, p-chloro(203Hg)mercuri-benzoic acid, N-ethyl(2,3-14C)maleimide and iode(1-14C)acetamide.

28. The method for measurement according to claim 20, wherein said metal compound and said metal composition are selected from the group consisting of gold colloid and iron-containing microbeads.