JPH07110331A - Human complement value measuring method and reagent component for measuring the value - Google Patents

Abstract

PURPOSE:To provide a human complement value measuring method which uses a stable liposome with a small difference between lots and capable of measurement in a short time and application for an automatic analyzer. CONSTITUTION:In this human complement value measuring method and reagent composition for the measurement therefor a liposome which includes a detectable marker substance and on which hapten is immobilized on the surface of a film, is used to cause a reaction of an antibody with a sample, the liposome and the hapten. The degree of destruction of a liposome film caused eventually is measured from the amount of emigration of the marker substance. Thus, in the method of measuring a human complement value, a calibration curve is prepared using serum derived from a rat, goat or sheep as complement standard liquid to determine the human complement value from the calibration curve.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring human complement value using liposomes and improvement of the reagent composition for the measurement.

[0002]

BACKGROUND OF THE INVENTION The complement system is a general term for about 20 kinds of proteins present in the serum of animals such as humans, and is important for biological defense such as recognizing and eliminating foreign substances and bacteria invading in the living body. Play a role. The complement system has two activation pathways, a classical pathway mainly activated by immune complexes and a second pathway activated by polysaccharides and the like. In the classical pathway, complement components are sequentially activated one after another, mainly by immune complexes of foreign substances (antigens) such as bacteria and antibodies against them, and finally the foreign substance membrane is destroyed and the foreign substance is killed or Dissolve. The reaction of erythrocytes sensitized with hemolysin to hemolysis and the reaction of hapten-sensitized liposome membrane destruction in the present invention are based on the classical pathway. On the other hand, the second pathway does not require the participation of antibodies, and is activated only by contact with, for example, a polysaccharide that is a component of the cell wall of bacteria or a virus.

In recent years, the activity of the complement system, that is, the measurement of complement value has been attracting attention as an index for diagnosis and treatment of acute glomerulonephritis, autoimmune diseases and the like. Currently, the complement titer is generally measured using Mayer's 50% hemolysis method and its modified method, in which the hemolytic activity of complement is measured using sheep red blood cells sensitized with hemolysin. Clinical Laboratory Law Recommendation, 1233-1234
Page, 29th edition, 5th edition, 1985, Kanehara Publishing Co., Ltd .; J.Clin.Che
m., 12 , 143 (1983), etc.]. For example, in the method described in “Clinical Test Method Recommendation”, first, a human serum sample is prepared by diluting each sample with gelatin veronal buffer in several stages. To this, sheep red blood cells sensitized with hemolysin prepared in advance were added, and the mixture was heated at 37 ° C for 60 minutes and then cooled to 2000 rpm.
After centrifuging at 10 minutes, the absorbance of the supernatant was adjusted to 541n using water as a control.
Measure in m. Based on this value, the degree of hemolysis of the serum sample at each dilution step is determined, then a graph is created, and the amount at which 50% hemolysis occurs (CH ₅₀ unit) is determined from this graph. However, in this method, it is necessary to prepare samples diluted to several kinds of dilution for the same sample, which causes an error in measurement. In addition, this is a complicated method in which the amount of 50% hemolysis is obtained from a graph. Further, sheep erythrocytes are used, but erythrocytes derived from living bodies are unstable, and susceptibility of erythrocytes to complement is different (difference between lots) due to individual difference of animals.

Therefore, instead of using erythrocytes, a method using more stable liposomes with less difference between lots has been developed, and a complement titer measuring method using liposomes prepared so as to easily undergo membrane damage reaction due to complement activity. Various reports have been made (JP-A-62-163966, JP-A-62-299764, JP-A-63-293470, JP-A-1-155271, etc.). However, even with the method using these liposomes, it is necessary to prepare samples diluted to several kinds of dilution for the same sample, and the amount of 50% liposomes to be dissolved is calculated from a graph, which is a complicated method. Complement value had to be measured. Further, in the method using liposomes and guinea pig serum having a known complement value or human serum having a known complement value, it is not necessary to prepare the same sample diluted to several kinds. However, when using guinea pig serum, there is a problem in that it takes a long time of 1 hour for measurement, and fresh human serum is difficult to obtain and manage.

On the other hand, in recent years, measurement using an automatic analyzer has been particularly emphasized, and it is desired to apply human complement number measurement to the automatic analyzer. However, since many automatic analyzers usually correspond to a reaction time of about 5 to 15 minutes, in the human complement titer measuring method that requires a long reaction time as in the conventional method, an automatic analyzer can be used. Measurement is difficult. Also, in humans and guinea pigs, the reaction rate at the start of the reaction is different in the case of a short reaction [J.
ods, 17 , 7-19 (1977)], there is a problem in using guinea pig serum as a standard solution for human complement value measurement. From the above, the development of a measurement method and a measurement reagent composition therefor, which uses liposomes that are more stable than living-body-derived erythrocytes and have a small difference between lots, is easy to operate, and can measure human complement value in a short time Was desired.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and is a method for measuring human complement value using liposomes which is stable and has a small lot-to-lot difference, and can be measured in a short time. Therefore, it is an object of the present invention to provide a method for measuring human complement value which can be applied to an automatic analyzer.

[0007]

Means for Solving the Problems The present invention uses a liposome in which a detectable labeling substance is encapsulated and a hapten is immobilized on the membrane surface, and a sample is reacted with the liposome and an antibody against the hapten, In the method of measuring human complement value by measuring the degree of destruction of the resulting liposome membrane from the amount of the labeled substance migrated, a calibration using serum derived from rat, goat or sheep as a complement standard solution It is an invention of the above-mentioned measuring method, characterized in that a line is created and the human complement value is determined from the calibration curve. Further, the present invention includes (i) a liposome in which a detectable labeling substance is encapsulated and a hapten is immobilized on the membrane surface, (ii) an antibody against the hapten,
ii) The invention is a reagent composition for human complement titer measurement, which comprises a combination of rat, goat or sheep-derived serum as a complement standard solution. That is, as a result of intensive studies to solve the above problems, the present inventors have found that it is difficult to obtain a complement value measuring method using a liposome in which a labeling substance is encapsulated and a hapten is immobilized on the membrane surface. Rat, goat or sheep serum can be used as a complement standard solution instead of human serum, and if a calibration curve is prepared using these serum,
They have found that the complement value can be measured in a short time, and have completed the present invention.

The present invention is characterized in that serum derived from rat, goat or sheep is used as a complement standard solution.
The strains of these animal species are not particularly limited. The sera of these animal species can of course be used as lyophilized products dissolved in water or an appropriate solution, and those sera diluted with an appropriate solution, ultrafiltration method, etc. As a matter of course, it is possible to use the one concentrated in 1., the serum from which components not involved in the complement reaction are removed, and the like. Further, it is optional to add sugars, proteins, preservatives, stabilizers, buffers and the like to these, if necessary.

The complement standard solution according to the present invention is mainly used for preparing a calibration curve, but it is of course possible to use it for other purposes if necessary. The liposome used in the present invention has a lipid bilayer structure obtained by dispersing a phospholipid or the like in an aqueous solution, whereby the inner water layer is separated from the outer water layer. It means artificial lipid vesicles. For example, J. liposome Res., 1 (3) , 339〜
377 (1989-90), or "Liposome Experiment Manual in Life Sciences", edited by Hiroshi Terada, Tetsuro Yoshimura, Springer Fairark Tokyo Co., Ltd., 1992, etc. However, any of these can be used in the present invention.

The main membrane constituents of the liposome used in the present invention include all those used as membrane constituents in ordinary liposome preparation,
There is no particular limitation. Chloroform extraction fractionation of sheep erythrocytes containing phospholipids [Biochemistry, 8 , 4149 (1969)]
Etc.], dimyristoylphosphatidylcholine (DMP
C), dipalmitoylphosphatidylcholine (DPP
C), distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, egg yolk phosphatidylglycerol,
Phospholipids such as dipalmitoylphosphatidylglycerol, dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, palmitoyloleoylphosphatidylcholine, ganglioside glycolipids, glycosphingolipids, glyceroglycolipids and other glycolipids, cholesterols ,
Natural lecithin (eg, egg yolk lecithin, soybean lecithin, etc.), or a mixture of one or two or more of them, which have a generally known composition, can be used.

The labeling substance to be encapsulated in the liposome is, for example, J. liposome Res., 1 (3) , 339-377 (1989 ) .
-90) etc., enzymes, coenzymes, water-soluble fluorescent substances, sugars, ionic compounds, chelate indicators, dyes,
Any substance such as a spin-label compound that can be detected by the membrane damage of the liposome may be used, and may be appropriately selected in consideration of the detection method, sensitivity and liposome stability. For example, alkaline phosphatase, glucose-6-
Enzymes such as phosphate dehydrogenase (G6PDH), β-galactosidase, horseradish peroxidase, asparaginase, urease, glucose oxidase, such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, etc. Coenzymes such as carboxyfluorescein, fluorescein isothiocyanate, fluorescein isocyanate, tetrarhodamine isothiocyanate, 5-dimethylamino-1-naphthalenesulfonyl chloride, and other fluorescent substances such as glucose and other sugars such as potassium dichromate and dichromic acid. Ionic compounds such as sodium and sodium chloride, eg Arsenazo III
Such as 4- (2-pyridylazo) resorcinol, 2- (5
-Bromo-2-pyridylazo) -5- (N-propyl-N-sulfopropylamino) phenol sodium salt and the like dyes, for example
Typical examples are spin-label compounds such as 2,2,6,6-tetramethylpiperidine-1-oxyl. Among these, in particular, when an enzyme is included, the reaction is amplified so that the sensitivity becomes high, and since it can be usually detected by measuring the absorbance, it can be applied to an automatic analyzer, which is preferable.

In the present invention, as a method for producing a liposome encapsulating a labeling substance, a conventionally known liposome preparation method can be used. For example, "Liposome Experiment Manual in Life Science", edited by Hiroshi Terada, Tetsuro Yoshimura, pp. 60-89, Springer Fairark Tokyo
Ltd., Vortex method shown in 1992, surfactant removal method,
In addition to French press method, ultrasonic treatment method, reverse phase evaporation method (R
EV method), ethanol injection method, ether injection method, pre-vesicle method, Ca ²⁺ fusion method, annealing
(Annealing) method, freeze-thaw fusion method, freeze-drying method, W / O / W emulsion method, SMGruner et al. [Biochemistr
y, 24 , 2833 (1985)] reported by Stable Plurilamell
All methods such as the ar Vesicle method (SPLV method) can be mentioned, and any of them can be used in the present invention. If necessary, the liposomes may be filtered to make the particle diameters uniform.

In the present invention, examples of haptens to be sensitized (immobilized) to liposomes include Forssmann antigen [Biochemistry, 8 , 4149 (1969)], GM ₁ [J. Immu.
nol.Methods, 85 , 53-63 (1985)] and other glycolipid antigens, dinitrophenyl [Biochemistry, Vol. 11, No. 22, 4085 (197).
2)] and trinitrophenyl [J.Immunol.Methods, 123 , 19
~ 24 (1989)], thyroid hormone thyroxine [Bio.Technology, April, 349 (1984)] phospholipid derivative, drug theophylline [J.Chem.Phar
m.Bull., 36 , 1086 (1988)] and phenytoin [Clin.Chem.,
38, 808 (1992)] phospholipid derivatives of the like, but incorporated into the liposome membrane may be at any as long as hapten antigen that reacts with antibodies, and is not particularly limited. The method for sensitizing the hapten to the surface of the liposome membrane may be carried out, for example, according to the literature relating to each of the above-mentioned haptens, or the cross-linking method known per se [“Zokusei Chemistry Experiment Course 5”, Immunobiochemistry Experiment Method, 1st edition, 1st edition, Japan Society for Biochemistry, Tokyo Kagaku Dojin, pages 144-148, March 14, 1986;
Biochemistry, 20 , 4229-4238 (1981); J. Biol. Biochem.,
257 , 286-288 (1982)] or the lipid activation method, etc., and other methods may of course be used.

The antibody used in the present invention may be any antibody as long as it is an antibody against the hapten sensitized to the liposome, and its origin is not particularly limited. For example, goat, rabbit, horse, sheep, those derived from mouse and the like, but among them, those that are unlikely to be affected by the rheumatoid factor and the like are preferable.
Examples include goat-derived antibodies and various monoclonal antibodies.

The method for measuring the amount of the labeling substance naturally varies depending on the type of the labeling substance used, but when the labeling substance is an enzyme, for example, "enzyme immunoassay", protein nucleic acid enzyme, separate volume No. 31, Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 51-63, Kyoritsu Shuppan Co., Ltd., September 10, 1987
The labeled substance may be measured according to the method described in, for example, when the labeled substance is a coenzyme, for example, US Pat.
The labeled substance may be measured according to the method described in the specification of 4,704,355. When the labeling substance is a fluorescent substance, the labeling substance is measured according to the method described in, for example, Illustrated fluorescent antibody, Akira Kawao, 1st edition, Soft Science Co., Ltd., 1983. If the labeling substance is a saccharide, the labeling substance may be measured according to the method described in, for example, Eur. J. Biochem., 21 , 80 (1971). When the labeling substance is an ionic compound, for example, Talamta, 31 , 37
(1984) and the like, the labeled substance may be measured according to the method described in (1984), and when the labeled substance is a dye, for example, Clin.C.
hem., 29 , 1587 (1983), etc., the labeled substance may be measured according to the method described above, and when the labeled substance is a spin labeling agent, for example, enzyme immunoassay, protein nucleic acid Enzymes, separate volume No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pp. 264-271, Kyoritsu Shuppan Co., Ltd., published September 10, 1987. The substance may be measured.

Examples of the buffer used in the measuring method of the present invention include phosphoric acid and its salts, boric acid and its salts, tris (hydroxymethyl) aminomethane (Tris),
Examples thereof include Good's Buffer, Veronal Buffer, etc., but are not limited to these, and any commonly used buffer can be used. The measuring reagents used in the measuring method of the present invention include proteins such as bovine serum albumin and gelatin, sugars, chelating agents, reducing agents, preservatives and other additives usually used in this field. However, it is optional to add them as necessary. The amount of various reagents used in the measuring method of the present invention,
The mass of the base used when the labeling substance is an enzyme, or the concentration range of the above-mentioned various additives, etc., which are added as necessary, is the concentration range usually used in the complement number measuring method known per se. It suffices to use appropriately selected.

The human complement titer measuring method of the present invention can be easily carried out, for example, as follows. First, a human serum sample, a liposome containing a labeling substance and having a hapten immobilized on the membrane surface, and an antibody against the hapten are reacted in an appropriate buffer solution, and the antibody and the hapten on the liposome membrane are bound. The antigen-antibody complex is made on the liposome membrane. As a result, the complement in serum is activated and damages the liposome membrane, and the liposome membrane is destroyed and the labeling substance contained in the liposome migrates from the liposome. Since this amount of migration reflects the complement titer in serum, the amount of this labeling substance that migrated is quantified. Then, the same procedure is performed using any one of rat, goat, and sheep-derived sera with known complement values as a complement standard solution in advance, and the human complement value is quantified from the obtained calibration curve. In this case, it suffices to appropriately select and perform the number (number) and the concentration of the complement standard solution to be used. The unit of human complement value is expressed as the CH ₅₀ value in the above-mentioned Mayer method, but the unit setting is not limited to this.

The assay method of the present invention comprises: (i) a liposome in which a detectable labeling substance is encapsulated and a hapten is immobilized on the membrane surface; (ii) an antibody against the hapten; and (iii) a complement standard solution. It can be carried out using a reagent composition which is combined with serum derived from rat, goat or sheep. Also, it goes without saying that, for example, when the labeling substance is an enzyme, its substrate and the like are also contained as one of its constituent components. The present invention is not limited to the method used, but can be sufficiently applied to a measurement system using an automatic analyzer, and the measurement can be performed easily and quickly. There are no particular restrictions on the combination of reagents and the like when performing measurement using an automatic analyzer, and the reagents and the like considered to be the best depending on the model or taking other factors into consideration. The combination may be appropriately selected and used.

Guinea pig serum, which has hitherto been used as a complement standard solution in a method for measuring complement value using liposomes, has a reaction rate at the start of reaction different from that of human serum.
It could not be used as a complement standard solution when carrying out the reaction in a short time of about 15 minutes. Further, when using fresh human serum as a complement standard solution, there is a problem that it is difficult to obtain and manage it. The present inventors have conducted extensive studies for a serum that is easily available and has a reaction rate at the initiation of the reaction that matches that of human serum.As a result, rat, goat and sheep serum are suitable for the purpose. It was found that there is such a thing, and by using these sera as a complement standard solution, the present invention has been reached in which human complement value can be quantified in a short time of about 5 to 15 minutes using liposomes. Hereinafter, the present invention will be described in more detail with reference to Experimental Examples and Examples, but the present invention is not limited thereto.

[0020]

【Example】

Experimental Example 1 (1) Preparation of G6PDH-encapsulated dinitrophenyl-sensitized liposome Glucose-6-phosphate dehydrogenase (abbreviated as G6PDH) was encapsulated and dinitrophenyl (hereinafter, referred to as DN) as a hapten.
Abbreviated as P. ) Is immobilized on the membrane as a liposome in the "Life Science Laboratory Manual"
(Hiroshi Terada, Tetsuro Yoshimura, pp. 60-89, Springer-Fairark Tokyo Co., Ltd., 1992). Dimyristoylphosphatidylcholine (DMPC) 71 μmol, Dimyristoylphosphatidylglycerol (DMPG) 8 μmol, Cholesterol 82
μmol and 0.8 μmol of phosphatidylethanolamine derivative of DNP were dissolved in 5 ml of chloroform and then dried under reduced pressure. To this, 7.5 ml of an aqueous solution of G6PDH (manufactured by Toyobo Co., Ltd.) [2,500 U / ml, 10 mM tris (hydroxymethyl) aminomethane (Tris) / HCl buffer (pH 7.8)] was added and mixed. The lipid hydration solution thus obtained is
The particles were sized through a membrane. The obtained liposome suspension was transferred to a centrifuge tube and centrifuged at 4 ° C. and 36,000 rpm,
Except for the enzyme not encapsulated in the liposome, 100m
The cells were suspended in M Tris / HCl buffer (pH 7.8) to obtain G6PDH-encapsulating DNP-sensitized liposomes (lipid concentration 5 nmol / ml).

(2) Comparison of liposome membrane-dissolving activity between human complement and various animal complements First, 10 μl of human serum of known complement value (35 CH ₅₀ U / ml) was added to
Gelatin veronal buffer solution (hereinafter abbreviated as GVB ⁺⁺ ) containing G6PDH-encapsulated DNP-sensitized liposomes (lipid concentration 5 nmol / ml) prepared in the above (1) (Maiya Inai et al., "Complementology") , Physic Drugs Publishing, 1983) 250 μl was added. After reacting at 37 ° C for 5 minutes, a sufficient amount of goat anti-DNP antibody and enzyme substrate [24 mM glucose-6-phosphate (G6P), 9 mM nicotinamide adenine dinucleotide (NAD)] were added and the pH was adjusted to 7.4. Add 125 μl of GVB ⁺⁺ prepared at 37 ° C
And reacted for 5 minutes. Complement liposome membrane dissolution (destruction)
The activity value of G6PDH migrated from the liposome by the action
Change in absorbance at 340 nm per minute by NADH (Δ
It was measured as A). Next, instead of human fresh serum, fresh serum of rat, goat, sheep, guinea pig, mouse, rabbit and hamster was used in the same manner, and the change in absorbance when human serum was used was compared. The results are shown in Table 1.

[0022]

[Table 1]

As is clear from Table 1, among the animal sera used, rat, goat and sheep sera had a liposome lytic activity (complement number 35 CH ₅₀ U / ml) equal to or higher than that of human serum. It was found to exhibit lytic activity. Then, rat, goat and sheep sera were respectively fed with human serum complement of 30 CH ₅₀ U.
When diluted with physiological saline so as to show an absorbance change (ΔA) corresponding to 1 ml / ml and the time course of the reaction was examined under the same measurement conditions as above, it was in good agreement with the case of human serum.

(3) Relationship between reaction time and liposomal membrane-dissolving activity Human, rat, goat, sheep, and guinea pig serum 10 μm each
250 μl of GVB ⁺⁺ containing G6PDH-encapsulated DNP-sensitized liposomes (lipid concentration 5 nmol / ml) prepared in (1) above was added to l, and 125 μl of GVB ⁺⁺ containing goat anti-DNP antibody was added at 37 ° C. Were reacted for 5, 10, 30, 60, 90 minutes. 125 μl of GVB ⁺⁺ containing an enzyme substrate (24 mM G6P, 9 mM NAD) and having a pH adjusted to 7.4 was added at each reaction time, and the mixture was reacted at 37 ° C. for 5 minutes. The activity value of G6PDH migrated from the liposome due to the action of complement to dissolve the liposome membrane was measured as a change in absorbance (ΔA) at 340 nm per minute by NADH. The results are shown in Table 2.

[0025]

[Table 2]

As is clear from Table 2, the changes in the absorbance of human, rat, goat and sheep serum became constant within 5 minutes, indicating that the reaction was almost completed. On the other hand, with guinea pig serum, it can be seen that it takes about 30 minutes for the absorbance change to become constant and the reaction to end. As is clear from these results, since rat, goat and sheep sera complete the reaction in a short time as in humans, rat, goat and sheep are used as the complement standard solution when measuring the human complement value. It can be seen that sheep serum is suitable.

Example 1. Measurement of human complement value using rat serum as complement standard solution Rat serum with known complement value (0, 15, 30, 38, 55 CH ₅₀ / m
l) 10 μl of G6PDH-encapsulating DNP prepared in (1) of Experimental Example 1
50 mM Tri containing sensitized liposomes (lipid concentration 5 nmol / ml)
250 μl of s / HCl buffer (containing 0.85% NaCl, pH 7.8) was added. After reacting at 37 ℃ for 5 minutes, a sufficient amount of goat anti-DNP
Contains antibody and enzyme substrate (24mM G6P, 9mM NAD)
50 mM Tris / HCl buffer (containing 0.85% NaCl, pH 7.8) 125
μl was added, and the mixture was reacted at 37 ° C. for 5 minutes. The activity value of G6PDH migrated from the liposome due to the action of complement to dissolve the liposome membrane was measured as the change in absorbance (ΔA) at 340 nm per minute by NADH to obtain a calibration curve. The results are shown in Fig. 1. Next, 15 human serum samples of unknown complement value were similarly operated, and the complement value was measured by the previously obtained calibration curve (FIG. 1). The results are shown in Table 3. In addition, for human serum of the same unknown complement number, a conventional complement number measuring method using sensitized sheep erythrocytes [One-point method: J. Clin. Chem., 1
2 , 143 (1983)], the complement value was measured. The results are also shown in Table 3.

[0028]

[Table 3]

As is apparent from Table 3, the measurement results of this method were in good agreement with those of the conventional method for measuring the complement number using sensitized sheep red blood cells.

Experimental Example 2. Preparation of phenytoin-sensitized liposome encapsulating G6PDH Encapsulating G6PDH and phenytoin (hereinafter referred to as hapten)
Abbreviated as PHT. ) Is immobilized on the membrane,
It was prepared in the same manner as (1) of Example 1. DMPC 71 μmo
l, DMPG 8 μmol, cholesterol 82 μmol, and
PHT phosphatidylethanolamine derivative 0.8μ
Mol was dissolved in 5 ml of chloroform and dried under reduced pressure.
To this, an aqueous solution of G6PDH (manufactured by Toyobo Co., Ltd.) [2,500 U / m
7.5 ml of 10 mM Tris / HCl buffer (pH 7.8)] was added and mixed. The lipid hydration solution thus obtained is
The particles were sized through a membrane. The obtained liposome suspension was transferred to a centrifuge tube and centrifuged at 4 ° C. and 36,000 rpm,
Except for the enzyme not encapsulated in the liposome, 100m
The cells were suspended in M Tris / HCl buffer (pH 7.8) to obtain G6PDH-encapsulating PHT-sensitized liposomes (lipid concentration 5 nmlo / ml).

Example 2. Measurement of human complement value using goat serum as a complement standard solution Goat serum with known complement value (0, 15, 35, _45, 60CH ₅₀ / ml) 1
To 0 μl, 250 μl of 50 mM Tris / HCl buffer solution (containing 0.85% NaCl, pH 7.8) containing the G6PDH-encapsulated PHT-sensitized liposome (lipid concentration 5 nmol / ml) prepared in Experimental Example 2 was added. 37 ° C
After reacting at room temperature for 5 minutes, 50mM T containing sufficient amount of rabbit anti-PHT antibody and enzyme substrate (24mM G6P, 9mM NAD)
125 μl of ris / HCl buffer (containing 0.85% NaCl, pH 7.8) was added, and the mixture was reacted at 37 ° C. for 5 minutes. The activity value of G6PDH migrated from the liposome due to the action of complement to dissolve the liposome membrane was measured as the change in absorbance (ΔA) at 340 nm per minute by NADH to obtain a calibration curve. The results are shown in Figure 2. Next, 15 human serum samples of unknown complement value were similarly operated, and the complement value was determined by the previously obtained calibration curve (FIG. 2). The results are shown in Table 4. In addition, a human serum with an unknown complement value is also used in a conventional complement quantification method using sensitized sheep erythrocytes [One-point method: J. Clin. Chem., 12 , 143].
(1983)], the complement value was measured. The results are also shown in Table 4.

[0032]

[Table 4]

As is clear from Table 4, the measurement results of this method were in good agreement with those of the conventional complement number measurement method using sensitized sheep red blood cells.

[0034]

INDUSTRIAL APPLICABILITY The present invention provides a rat and goat whose reaction rate at the start of the reaction is the same as that of human serum as a complement standard solution for preparing a calibration curve in a method for measuring human complement value using liposomes. Alternatively, by using sheep serum, the measurement can be easily performed in a short time of about 5 to 15 minutes, and measurement using an automatic analyzer is also possible, which is more accurate than conventional methods. In addition, it is possible to measure a large amount of samples with good reproducibility, which is a remarkable effect.

[0035]

[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 1, in which the horizontal axis represents the complement value (CH ₅₀ / ml) of rat serum,
The vertical axis represents the change in absorbance at 340 nm per minute (Δ
Represent A) respectively.

[Fig. 2] Fig. 2 shows a calibration curve obtained in Example 2, in which the abscissa represents the complement value (CH ₅₀ / ml) of goat serum and the ordinate represents 1 minute at 340 nm. Absorbance change (ΔA)
Respectively.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 21, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0015

[Correction method] Change

[Correction content]

The method for measuring the amount of the labeling substance naturally varies depending on the type of the labeling substance used. For example, when the labeling substance is an enzyme, for example, "enzyme immunoassay", protein
Nucleic Acid Enzymes, Separate Volume No31, Tsunehiro Kitagawa / Toshio Minamihara / Akio Tsuji / Eiji Ishikawa, 51-63, Kyoritsu Shuppan Co., Ltd., 1
The labeled substance may be measured according to the method described in, for example, September 10, 987. When the labeled substance is a coenzyme, for example, see US Pat. No. 4,704,355. The labeled substance may be measured according to the method described. When the labeling substance is a fluorescent substance, for example, Illustrated fluorescent antibody, Akira Kawao. First edition. The labeled substance may be measured according to the method described in Soft Science Co., Ltd., 1983, etc. When the labeled substance is a saccharide,
For example, Eur. J. Biochem. , 21 , 80 (1
The labeled substance may be measured according to the method described in 971) or the like. When the labeling substance is an ionic compound,
For example, the labeling substance may be measured according to the method described in Talanta , 31 , 375 (1984) or the like. When the labeling substance is a dye, for example, Clin. Ch
em. , 29 , 1587 (1983), etc., and the labeled substance may be measured according to the method described in, for example, when the labeled substance is a spin labeling agent, for example, enzyme immunoassay, protein nucleic acid enzyme, Separate Volume No. 31, Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 264-271
Page, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987, etc., and the labeled substance may be measured.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

[0020]

Examples Experimental Example 1 1) Preparation of G6PDH-encapsulated dinitrophenyl-sensitized liposome Glucose-6-phosphate dehydrogenase (abbreviated as G6PDH) is encapsulated and dinitrophenyl (hereinafter referred to as DNP) as a hapten. ) Is immobilized on the membrane in a "Liposome Experiment Manual in Life Science" (edited by Hiroshi Terada, Tetsuro Yoshimura, pp. 60-89, Springer Fairark Tokyo Co., Ltd., 1992).
Prepared according to the Vortex method described in. Dimyristoylphosphatidylcholine (DMPC) 71μmo
l, Dimyristoylphosphatidylglycerol (DM
PG) 8 μmol, cholesterol 82 μmol, and phosphatidylethanolamine derivative of DNP 0.8
After dissolving μmol in 5 ml of chloroform, it was dried under reduced pressure. An aqueous solution of G6PDH (manufactured by Toyobo Co., Ltd.) [2,500 U / ml, 10 mM tris (hydroxymethyl) aminomethane (Tris) / HCl buffer (p
H7.8)] 7.5 ml was added and mixed. The lipid hydration solution thus obtained was sized through a 0.2 μm membrane. The obtained liposome suspension was transferred to a centrifuge tube and centrifuged at 4 ° C. and 36,000 rpm to remove the enzyme not encapsulated in the liposome and finally to 100 mM.
Suspend in Tris / HCl buffer (pH 7.8),
A DNP-sensitized liposome encapsulating G6PDH was obtained.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

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Example 1. Measurement of human complement value using rat serum as a complement standard solution Rat serum with known complement value (0, 15, 30, 38, 55
₅₀ mM Tris / HCl containing 10 μl of CH ₅₀ U / ml ) containing G6PDH-encapsulated DNP-sensitized liposomes (lipid concentration 5 nmol / ml) prepared in (1) of Experimental Example 1.
Buffer solution (containing 0.85% NaCl, pH 7.8) 250
μl was added. After reacting at 37 ° C. for 5 minutes, a sufficient amount of goat anti-DNP antibody and enzyme substrate (24 mM G6P, 9 mM) was added.
125 μl of 50 mM Tris / HCl buffer (containing 0.85% NaCl, pH 7.8) containing mM NAD)
Was added and reacted at 37 ° C. for 5 minutes. G6PDH migrated from the liposome due to the action of complement to dissolve the liposome membrane
Activity value of 340 nm per minute by NADH
Was measured as a change in absorbance (ΔA) to obtain a calibration curve. The results are shown in Fig. 1. Next, 15 human serum samples of unknown complement value were similarly operated, and the complement value was measured by the previously obtained calibration curve (FIG. 1). The results are shown in Table 3. In addition, the same human serum of unknown complement number was used for a conventional complement number assay method using sensitized sheep red blood cells [One-point method: J. Cl
in. Chem. , 12 , 143 (1983)], the complement value was measured. The results are also shown in Table 3.

[Procedure amendment 4]

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[Correction target item name] 0028

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[0028]

[Table 3]

[Procedure Amendment 5]

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[Name of item to be corrected] 0030

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Experimental Example 2. Preparation of phenytoin-sensitized liposome encapsulating G6PDH A liposome in which phenytoin (hereinafter abbreviated as PHT) as a hapten was immobilized on a membrane was prepared in the same manner as in Example 1 (1). DMPC
71 μmol, DMPG 8 μmol, cholesterol 82 μmol, and PHT phosphatidylethanolamine derivative 0.8 μmol were added to chloroform 5 m.
After dissolving in 1 l, it was dried under reduced pressure. An aqueous solution of G6PDH (manufactured by Toyobo Co., Ltd.) [2,500 U / ml, 10
mM Tris / HCl buffer (pH 7.8)] 7.5
ml was added and mixed. The lipid hydration solution thus obtained was sized through a 0.2 μm membrane. The obtained liposome suspension was transferred to a centrifuge tube, and kept at 4 ° C for 36.0
Centrifuge at 00 rpm to remove the enzyme not encapsulated in the liposome, and finally to 100 mM Tris / HCl.
G6PDH-encapsulating P by suspending in a buffer solution (pH 7.8)
HT-sensitized liposomes were obtained.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

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Example 2. Measurement of human complement value using goat serum as a complement standard solution Goat serum with known complement value (0, 15, 35, 45, 60 C
H ₅₀ U / ml ) 10 μl of G6 prepared in Experimental Example 2
PDH-encapsulated PHT-sensitized liposomes (lipid concentration 5 nmol
/ Ml) in 50 mM Tris / HCl buffer (containing 0.85% NaCl, pH 7.8) (250 μl) was added. After reacting at 37 ° C for 5 minutes, a sufficient amount of rabbit anti-PHT antibody and enzyme substrate (24 mM G6P, 9 mM
125 μl of 50 mM Tris / HCl buffer (containing 0.85% NaCl, pH 7.8) containing NAD) was added, and the mixture was reacted at 37 ° C. for 5 minutes. The activity value of G6PDH migrated from the liposome due to the action of complement to dissolve the liposome membrane was measured as a change in 34 Onm absorbance (ΔA) per minute by NADH to obtain a calibration curve. The results are shown in Figure 2. Next, 15 human serum samples of unknown complement value were similarly operated, and the complement value was determined by the previously obtained calibration curve (FIG. 2). The results are shown in Table 4. In addition, the same human serum of unknown complement number was used for a conventional complement number assay method using sensitized sheep red blood cells [One-point method: J. Cli
n. Chem. , 12 , 143 (1983)], the complement value was measured. The results are also shown in Table 4.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

[0032]

[Table 4]

[Procedure Amendment 8]

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[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

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[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 1, in which the abscissa represents the complement value of rat serum ( CH ₅₀ U / ml ).
Also, the vertical axis represents the change in absorbance (ΔA) per minute at 340 nm.

FIG. 2 shows the calibration curve obtained in Example 2, in which the abscissa represents the complement value ( CH ₅₀ U / ml ) of goat serum,
The vertical axis represents the change in absorbance (ΔA) per minute at 340 nm.

[Procedure Amendment 9]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

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[Figure 1]

[Procedure Amendment 10]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

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[Fig. 2]

Claims (4)

[Claims] 1. A liposome in which a hapten is immobilized on the surface of a membrane containing a detectable labeling substance, and a sample, the liposome, and an antibody against the hapten are reacted with each other, resulting in destruction of the liposome membrane. In the method for measuring human complement value by measuring the degree from the amount of labeled substance, a calibration curve is prepared using serum derived from rat, goat or sheep as a complement standard solution, and the calibration curve is obtained. The human complement value is determined from the above-mentioned measurement method. 2. The measuring method according to claim 1, wherein the labeling substance is an enzyme. 3. (i) A liposome containing a detectable labeling substance and having a hapten immobilized on the membrane surface, (ii) an antibody against the hapten, (iii) a rat, goat or a complement standard solution. A reagent composition for measuring human complement value, which is formed by combining with serum derived from sheep. 4. The reagent composition according to claim 3, wherein the labeling substance is an enzyme, and the substrate component of the enzyme is further combined.