Immunoassay process and liquid reagents used therefor

Abstract: A combination of reagents comprising (i) a first liquid containing (a) microcapsules fixing an analyte to be measured on surfaces thereof and containing a labeled substance therein and (b) microcapsules encapsulating an antibody and having different capsule walls from the microcapsules (a) on lysing property for a capsule wall lysing, and (ii) a second liquid containing a complement is suitable for immunoassay based on complement-dependent immune lysis of microcapsules.

Description: BACKGROUND OF THE INVENTION The present invention relates to a modification of an immunoassay based on complement-dependent immune lysis of microcapsules and provides liquid reagents used therefor which have been stabilized and can be used in autoanalyzers. Immunoassay is a measuring method utilizing an antigen-antibody reaction and is widely used, for example, as a method for measuring trace components in body fluids specifically. As immunoassays which are now generally often used, there may be exemplified radioimmunoassay (RIA), and enzyme immunoassay (EIA). These methods permit quantitative measurement of trace components in samples but involve individual problems. That is, RIA is disadvantageous, for example, in that since isotopes are used therein, RIA requires special facilities and problems arise with disposal of waste. EIA is disadvantageous, for example, in that it requires a relatively long measurement time and the EIA process is difficult to apply to an autoanalyzer. Therefore, as an immunoassay involving none of these problems, there has recently been proposed and noted an immunoassay based on complement-dependent immune lysis of microcapsules. A typical example of this method is a method using microcapsules (e.g. lipid film vehicles (liposomes)) involving fixing an analyte to be measured on surfaces thereof said microcapsules containing a labelled substance (e.g. enzyme) therein (hereinafter abbreviated as "labeled microcapsules") (hereinafter abbreviated as "immunoassay method based on complement-dependent immune lysis of microcapsules") (Japanese Patent Appln. Kokai No. 56-132564, U.S.P. 4,342,826). This method is as follows. A sample, labeled microcapsules, and an antibody to an analyte to be measured are fixed to carry out the antigen-antibody reaction, after which a complement is added. The complement is activated by an antigen-antibody complex formed on the surfaces of the labeled microcapsules and lyses the membrane walls of the microcapsules to liberate a labeled substance from the labeled microcapsules. The amount of analyte to be measured in the sample is determined on the basis of the amount of the labeled substance liberated. This immunoassay method is based on complement-dependent immune lysis of microcapsules introduced by an antigen-antibody reaction and hence makes it possible not only to measure a slight amount of an analyte to be measured, specifically, but also to carry out a series of reactions in a uniform reaction system. Therefore, it permits simpler and rapider measurement than do conventional methods such as RIA and EIA. Accordingly, it is noted. This method is, however, difficult to apply to an autoanalyzer, in particular, an autoanalyzer using two liquid reagents which is now a leading autoanalyzer, and substantially no case has been reported for the application of the method to such an autoanalyzer. The cause of this is that when the reagents (labeled microcapsules, antibody to an analyte to be measured, and complement) used in said method are stored in a proper combination in two liquid reagents, the stability of the liquid reagents is not sufficient. When the three main reagents used in said method are divided between two liquid reagents, and stored therein, the following three combinations are presented: a) a combination of a mixed solution of complement and labelled microcapsules, and separately an antibody solution, b) a combination of a mixed solution of complement and antibody, and separately a solution of labelled microcapsules, c) a combination of a mixed solution of labelled microcapsules, and an antibody, and separately a complement solution. Whichever combination specified above is employed for preparing the liquid reagents, the resulting liquid reagents have a low stability during storage and hence are of no practical use. For example, in the case of the combination of a), the complement is inactivated by the interaction between the labeled microcapsules and the complement which coexist in the liquid reagent, so that immunalysis is gradually reduced. In the case of the combination of b), when an analyte to be measured is a substance, for example a drug, which is unlikely to be mixed in a solution of complement derived from an animal, the resulting liquid reagent is stable to a certain extent. But when the analyte to be measured is a substance which is very likely to exist in the solution of complement, for example, a hormone or a tumour marker, the antigen-antibody reaction and inactivation of the complement occur in the mixed solution of the complement and the antibody, so that the stability of this liquid reagent is lowered. In the case of the combination of c), an analyte to be measured (an antigen) fixed on the labeled microcapsules reacts with the antibody previous to the actual quantitative determination resulting in lowering of the measurement sensitivity. Therefore, depending on the analyte to be measured, there is now often no choice but to use three liquid reagents for an immunoassay method based on complement-dependent immune lysis of microcapsules. Accordingly, there has been an eager desire for the advent of liquid reagents for immunoassay methods based on complement-dependent immune lysis of microcapsules and using two liquid reagents which can be used in autoanalyzers. SUMMARY OF THE INVENTION The present invention was made in consideration of such conditions and is intended to provide an immunoassay method based on complement-dependent immune lysis of microcapsules and using two liquid reagents, said liquid reagents having high stability during storage, and being applicable to autoanalyzers. The present invention provides a liquid reagent used for an immunoassay method based on complement-dependent immune lysis of microcapsules comprising: (a) first microcapsules having an analyte to be measured immobilized on surfaces thereof and containing a substance capable of acting as a label therein, wherein said first microcapsules are capable of being lysed by complement once activated by an antigen-antibody complex formed on the said surfaces, and (b) second microcapsules having different capsule walls from said first microcapsules and encapsulating an antibody specific to said analyte, wherein the capsule walls of said second microcapsules are more easily lysed by a second substance having a cell membrane lysing activity than the capsule walls of said first microcapsules. The present invention also provides an immunoassay reagent combination used for an immunoassay method based on complement-dependent immune lysis of microcapsules comprising: (a) a first liquid comprising: (i) first microcapsules having an analyte to be measured immobilized on surfaces thereof and containing a substance capable to act as a label therein, wherein said first microcapsules are capable of being lysed by complement once activated by an antigen-antibody complex formed on the said surfaces, and (ii) second microcapsules having different capsule walls from said first microcapsules and encapsulating an antibody specific to said analyte, wherein the capsule walls of said second microcapsules are more easily lysed by a second substance having a cell membrane lysing activity than the capsule walls of said first microcapsules. (b) a second liquid containing complement. The present invention further provides an immunoassay method based on complement-dependent immune lysis of microcapsules comprising: (a) a first step of reacting a sample containing an analyte to be measured and a substance other than the complement to be used when carrying out the second step, said substance having a cell membrane lysing activity, with a first liquid comprising: (i) first microcapsules having an analyte immobilized on surfaces thereof and containing a substance capable to act as a label therein, wherein said first microcapsules are capable of being lysed by complement once activated by an antigen-antibody complex formed on the said surfaces, and (ii) second microcapsules having different capsule walls from said first microcapsules and encapsulating an antibody specific to said analyte, wherein the capsule walls of said second microcapsules are more easily lysed by the said substance having a cell membrane lysing activity than the capsule walls of said first microcapsules, wherein the said substance is present in a concentration sufficient to lyse said second microcapsules but not said first microcapsules. (b) a second step of reacting the resulting reaction solution with a second liquid containing complement, and (c) a third step of measuring the amount of the substance capable of acting as a label liberated by the second step. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a calibration curve for T4 obtained in Example 1. Fig. 2 shows a correlational graph prepared on the basis of measured values obtained by Example 3 and Comparative Example 1. DESCRIPTION OF THE PREFERRED EMBODIMENTS Various embodiments of the subject invention are summarised above. In the course of earnest study for finding an immunoassay method based on complement-dependent immune lysis of microcapsules which is applicable to autoanalyzers, we, the present inventors, found that the microcapsule lysing properties of various substances having - cell membrane lysing activity (i.e. "capsule wall lysin") vary depending on the constituents of the microcapsule wall and its preparation method. We noticed this point and further investigated. Consequently, we found that an immunoassay method based on complement-dependent immune lysis of microcapsules and using two liquid reagents which have a high stability during storage and can be used in antoanalyzer, is possible using an antibody used in immunoassay methods after encapsulating the antibody in microcapsules which are more easily lysed by a specific capsule wall lysin, i.e., a surfactant, than labeled microcapsules. Thus, we accomplished the present invention. That is, the liquid reagent combination of this invention comprises a first liquid comprising labelled microcapsules and microcapsules characterized by encapsulating an antibody (hereinafter abbreviated as "encapsulated antibody"), and a second liquid containing a complement. As the specific capsule wall lysin used in the measuring process of this invention, surfactants can be exemplified. The capsule wall lysin is not critical so long as it lyses the capsule wall of encapsulated antibody but not the capsule wall of labeled microcapsules, and so long as it does not affect the measurement. As such surfactants, the following can be exemplified. Nonionic surfactants, for example, polyoxyethylene alkyl ethers such as polyoxyethylene cetyl ether, and polyoxyethylene lauryl ether; polyoxyethylene alkylphenyl ethers such as polyoxyethylene otylphenyl ether; polyoxyethylene alkyl esters such as polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitatte, polyoxyethylene sorbitan monostearate, and polyoxyethylene sorbitan triolate; methylglucamide derivatives such as octanoyl-N-methylglucamide, nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide; and alkyl sugar derivatives such as n-octyl-β-D-glucoside; Anionic surfactants, for example, sodium dodecyl sulfate (SDS), laurylbenzenesulfonic acid, deoxycholic acid, cholic acid and tris(hydroxymethyl)aminomethane dodecylsulfite (Tris DS); Cationic surfactants, for example, alkylamine salts such as octadecylamine acetic acid salt, tetradecylamine acetic acid salt, stearylamine acetic acid salt, laurylamine acetic acid salt and lauryldiethyanolamine acetic acid salt; quaternary ammonium salts such as octadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, lauryltrimethylammonium chloride, allyltrimethylammonium methylsufate, benzalkonium chloride, tetradecyldimethylbenzylammonium chloride, octadecyldimethylbenzylammonium chloride and lauryldimethylbenzylammonium chloride; and alkylpyridinium salts such as laurylpyridinium chloride and stearylamidomethylpyridinium chloride; Amphoteric surfactants, for example, 3-[(3-cholamidoamidopropyl)dimethylammonio]-1-propane sulfonate and 3-[(3- 3-[(3-cholamidoamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate; Natural surfactants, for example, saponin (derived from soybean) and digitonin. The concentration of these surfactants is not critical so long as they dissolve the capsule wall of the encapsulated antibody but not the capsule wall of the labelled microcapsule. The concentration of the surfactants at the time of measurement is preferably chosen usually in the range of 0.01 to 2 V/V%, preferably 0.1 to 1 V/V%. In this invention, there cannot be used complement, polymyxin B and mellitin instead of the above mentioned surfactant which are conventionally used as capsule wall lysins for microcapsules such as liposome. Although the reason for this is not clear, it is conjectured, as follows. The liposome membrane lysis by a capsule wall lysin such as complement, does not destroy the structure of the liposome itself completely but merely makes a hole in a part of the membrane. Therefore, low-molecular-weight substances (e.g. substances which can emit fluorescence and substrates for enzymes) can go in and out of the liposome freely through the hole, but high-molecular-weight substances such as antibody cannot pass through the hole and are kept in the liposome. As a microcapsule for the encapsulated antibody used in the measuring process of this invention, any microcapsule can be exemplified without particular restriction so long as it is more easily lysed by the surfactant than the labelled microcapsule, though liposomes having such a property are preferred because of ease of preparation. As a method for preparing such liposomes, there can be exemplified well-known methods such as the voltexing method, sonication method, surfactant removal method, reverse-phase evaporation method (REV method), ethanol infusion method, ether infusion method, pre-vesicle method, French press extrusion method, Ca²⁺ fusion method, annealing method, freeze thawing method, freeze drying method and the W/O/W emulsion method, and methods such as the stable plurilamellar vesicle method (SPLV method) recently reported by S.M. Gruner et al. [Biochemistry, 24 , 2833 (1985)] as well as the method using a lipopolysaccharide as one constituent of the membrane which has been reported by some of the present inventors (Japanese Patent Appln. Kokai (Laid-Open) No. 63-107742). Of these, particularly preferable are methods which permit preparation of unilamellar liposomes, for example, the surfactant removal method and the sonication method. As the main constituent of the membrane of the liposome, there can be exemplified each or combinations of two or more of substances used as materials for membranes in preparation of conventional liposomes, i.e., natural lecithins (e.g. egg yolk lecithin and soybean lecithin) and phospholipids such as dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPS), dimyrictoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidic acid (DMPA) and egg yolk phosphatidylglycerol; mixtures of these substances and cholesterols; and combinations of the mixtures and polysaccharides. Particularly preferable examples of the main constituent of the membrane are lipids having a low Tc (eg. egg yolk lecithin) and lipids which tend to be in a hexagonal phase (eg. phosphatidylethanol-amine and acidic lipids). The antibody contained in the microcapsule in the encapsulated antibody of this invention is not critical, and any antibody may be used so long as it is an antibody to an analyte to be measured. That is, there may be used either polyclonal antibodies prepared by immunizing animals such as horse, cattle, sheep, rabbit, goat, rat and mouse etc. with an analyte to be measured, according to a conventional method. Such conventional methods are for example, any of the methods described in Tadashi Matsuhashi et al. "Meneki Jikken-gaku Nyumon" 2nd. ed., GAKKAI-SHUPPAN CENTER Ltd., 1981; and E. Harlow et al. "Antibodies" Cold Spring Harbor Laboratory, 1988, pp.53-138, or monoclonal antibodies produced by Hybridomas obtained by fusing cells from a tumor line of mouse together with mouse spleen cells previously immunized with an analyte to be measured, according to the conventional method, i.e., the cell fusion method established by G. Köhler and C. Milstein (Nature, 256, 495, 1975). These polyclonal and/or monoclonal antibodies may be used singly or in a combination of two or more thereof. Needless to say, they may be used, if necessary, after digesting them with an enzyme such as pepsin or papain into F(ab')₂, Fab' or Fab. A method for preparing the encapsulated antibody of this invention is explained below in detail taking the case of the preparation of liposomes by the surfactant removal method. First, such phospholipids and cholesterols described above are dissolved in a suitable organic solvent (e.g. chloroform, an ether, an alcohol, and the resulting solution is concentrated to dryness under reduced pressure and then sufficiently dried under reduced pressure in a desiccator. Subsequently, an aqueous surfactant solution (20 to 100 mM) is added to the lipid film thus formed and the film is uniformly dispersed thereinto. The surfactant used here includes, for example, those heretofore often used in the art, such as cholic acid, polyoxyethylene octylphenyl ether and octyl glucoside, though surfactants having a high critical micelle concentration (CMC), such as octyl glucoside are preferred. Next, if necessary, a lipopolysaccharide is added in powder form as it is or in solution, followed by adding thereto a solution of the desired antibody to be encapsulated (usually a solution having a concentration of 0.1 to 20 mgAb/ml, preferably 1 to 10 mgAb/ml), and the resulting mixture is sufficiently stirred. It is most preferable to remove the surfactant immediately after the stirring, and suitable methods for the removal includes per se well-known methods such as dialysis, gel filtration and adsorption on resin. As to the treatment conditions, the treatment time is 1 to 24 hours, and the treatment temperature may be chosen in the range of 0° to 70°C though it is somewhat varied depending on the constituents of membrane of the liposome. For removing the surfactant, gel filtration through Sepharose 4B (a trade name, Pharmacia AB) and centrifugation, are particularly advantageous because free antibody can also be removed at the same time. The liposomes thus obtained are used or stored after being concentrated by ultrafiltration so as to have a predetermined concentration. For making the sizes of the liposomes uniform, a method using a generally used polycarbonate membrane may be employed, though gel filtration (using, for example, Sephacryl S-1000 (a trade name, Pharmacia AB)) is also effective. Also when the encapsulated antibody of this invention is prepared by a method other than the surfactant removal method, it is sufficient that it is prepared according to any per se well-known method or other method for preparing liposomes. As the labeled microcapsule used in the measuring process of this invention, any microcapsule can be exemplified without particular restriction so long as it is lysed with more difficulty by the surfactant than the encapsulated antibody. Usually, a liposome having such a property is actually chosen because of ease of preparation. As a method for preparing such liposomes, there can be exemplified all of the per se well-known preparation methods of liposomes previously exemplified for the microcapsule for the encapsulated antibody. As the constituents of the membrane, there can be exemplified all of the per se well-known materials for the membrane previously exemplified for the microcapsule for the encapsulated antibody. In particular, liposomes prepared including as a constituent of the membrane a lipid with a high phase transition temperature (e.g. DPPC, DSPC) by a method which permits preparation of multilamellar liposomes (e.g. SPLV method, REV method), are more preferable because they are lysed with more difficulty by the surfactant than liposomes prepared from other methods and/or materials. Liposomes containing a labeled substance therein are thus prepared and an analyte to be measured is fixed onto their surfaces by a conventional method. As such a conventional method, when a water-soluble protein is fixed, there can be used, for example, the crosslinking method, the lipid activation method, and the method using a lipopolysaccharide disclosed in Japanese Patent Appln. Kokai (Laid-Open) No. 63-107742. When a low-molecular-weight hapten is fixed, there can be used, for example, a method which comprises converting the hapten to a lipid derivative previously and then incorporating the liquid derivative into liposomes. Thus, the labeled microcapsules of this invention can easily be obtained. As the labeled substance to be contained in the labeled microcapsules, any labeled substance can be exemplified without particular restriction so long as it is a detectable labeled substance usually used in immunoassay method based on complement-dependent immune lysis of microcapsules. Typical examples of the labeled substance are enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase and β-galactosidase; substances which can emit fluorescent, such as carboxyfluorescein; dyes such as Arsenazo III, 4-(2-pyridylazo)resorcinol and 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol sodium salt; luminescent substances such as luminol, bis(2,4,6-trichlorophenyl) oxalate and N-methylacridinium ester; and substances having spin properties, which are represented, for example, by 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO). As the complement used in the measuring process of this invention, there can be exemplified, without exception, all the complements usually used in the art, for example, complements derived from blood of animals such as human being, guinea pig, horse and sheep, which have been properly purified by any conventional method. It is sufficient that the concentration ranges of the reagents used in the measuring process of this invention and various antiseptics optionally added in said process are properly selected from the concentration ranges which are usually employed for per se well-known reagents for immunoassay methods based on complement-dependent immune lysis of microcapsules. Buffers used in the measuring process of this invention include, for example, tris(hydroxymethyl)aminomethane, Good's buffers and Veronal, but are not limited thereto. The reagent combination of this invention used for the measuring process of this invention is characterized by comprising the following two liquids. First liquid: comprising encapsulated antibody, labeled microcapsules, and if necessary, a buffer and any substances necessary to enable the "label" substance to act as such (e.g. coenzyme, substrate for enzyme) an antiseptic. Second liquid: containing a complement and if necessary, a substrate, coenzyme, buffer, antiseptic. As to the pH's of the reagent combination of this invention, the pH of the first liquid is chosen usually in the range of 6 to 9, preferably 7 to 8, and the pH of the second liquid is chosen usually in the range of 6 to 9, preferably 7 to 8. The pH's are adjusted using, for example, the buffers exemplified above. The measuring process of this invention using the reagent combination of this invention is practiced, for example, as follows. First, a surfactant capable of lysing the capsule wall of the microcapsule containing the encapsulated antibody but not the capsule wall of the labeled microcapsule is added to the first liquid together with a sample containing an analyte to be measured, to liberate the antibody. Then, the second liquid containing a complement is allowed to act on the resulting mixture, followed by measurement by a conventional method. It is sufficient that the measurement itself is carried out according to a per se well-known immunoassay method based on complement-dependent immune lysis of microcapsules. More specifically, first, a specimen for measurement is prepared by mixing a sample containing an analyte to be measured with a suitable surfactant. The specimen and the first liquid are mixed and then reacted with each other usually at 20° to 50°C, preferably 25° to 40°C, for 5 minutes to 1 hour. Subsequently, the resulting reaction solution is mixed with the second liquid, and the resulting mixture is subjected to reaction usually at 20° to 50°C, preferably 25° to 40°C, for 5 minutes to 1 hour. Then, the amount of the label substance liberated from the labeled microcapsules is measured by a per se well-known measuring method suitable to the properties of the "label" substance. The concentration of the analyte to be measured in the sample is determined on the basis of the thus obtained amount of the label substance by using a calibration curve showing the relationship between the concentration of the analyte to be measured and the amount of the label substance liberated from the labelled microcapsules, said calibration curve having been previously prepared by subjecting several samples containing known concentrations of the analyte to be measured to the same procedure as described above. In the above procedure, the order of reacting the first liquid and the second liquid with the specimen should not be changed. After mixing the specimen with the first liquid, they should be reacted with each other for a certain time. This is because measurement of the analyte to be measured becomes practically impossible when the first liquid and the second liquid are added in reverse order and reacted with the specimen, or when the first liquid and the second liquid are added at substantially the same time and reacted with the specimen. As a method for measuring the amount of the label substance, the following methods can be exemplified. For example, when the label substance is an enzyme, it is measured according to, for example, any of the methods described, for instance, in Tsunehiko Kitagawa, Toshio Nanbara, Akio Tsuji, and Eiji Ishikawa "Koso Men-eki Sokuteiho", an extra issue No. 31 of Tanpakushitsu Kakusan Koso, pp. 51-63, KYORITSU-SHUPPAN Ltd., published on Sept. 10, 1987; and E. Harlow et al. "Antibodies" Cold Spring Harbor Laboratory, 1988, pp. 592-598. When the label substance is a substance which can emit fluorescence, it is measured according to, for example, any of the methods described in Akira Kawano "Zusetsu Keikokotai" 1st ed. Soft Science, Inc., 1983; and R. M. Nakamura et al. "Immunoassays" Alan R. Liss, Inc., New York, 1980, pp. 10. When the label substance is a luminescent substance, it is measured according to, for example, any of the methods described in Tsunehiro Kitazawa, Toshio Nanbara, Akio Tsuji and Eiji Ishikawa "Koso Men-eki Sokuteiho", an extra issue No. 31 of Tanpakushitsu Kakusan Koso, pp. 252-263, KYORITSU-shuppan Ltd., published on Sept. 10, 1987; and R.M. Nakamura et al. "Immunoassays" Alan R. Liss, Inc., New York, 1980, pp. 174-176. When the label substance is a substance having spin properties, it is measured according to, for example, any of the methods described in Tsunehiro Kitagawa, Toshio Nanbara, Akio Tsuji and Eiji Ishikawa "Koso Men-eki Sokuteiho" an extra issue No. 31 of Tanpakushitsu Kakusan Koso, pp. 264-271, KYORITSU-SHUPPAN Ltd., published on Sept. 10, 1987; and R.M. Nakamura et al. "Immunoassays" Alan R. Liss. Inc., New York, 1980, pp. 213-218. As an analyte which can be measured by the measuring process of this invention, there can be exemplified, without particular restriction, any substance which permits preparation of an antibody thereto, or any antibody produced in a living body. Typical examples of the analyte to be measured include proteins, lipids, hormones, drugs and specific substances, which are contained in body fluids such as serum, blood, plasma and urine. More specific examples of the analyte to be measured include tumor markers such as α-fetoprotein (AFP), CA19-9, prostate gland specific antigen (PSA), and carcinoembryonic antigen (CEA); hormones such as insulin, human chorionic gonadotropin (hCG), thyroxine (T4), triiodothyronine (T3), prolactin, thyroid stimulating hormone (TSH), and the like; drugs such as digoxin, phenytoin, morphine and nicotine; and antibodies such as anti-Toxoplasma antibody. This invention is illustrated below in further detail with reference to Examples, which are not by way of limitation but by way of illustration. EXAMPLES Referential Example 1 Preparation of encapsulated antibody In a round bottom flask were placed 2 ml of a 20 mM solution of egg yolk lecithin in chloroform and 2 ml of a 20 mM solution of cholesterol in chloroform, and mixed. Then, the solvent was distilled off by means of a rotary evaporator, followed by drying under reduced pressure in a desiccator for about 2 hours, whereby a lipid thin film was formed on the interior surface of the flask. To the lipid thin film were added 0.6 ml of a 200 mM aqueous n-octyl glucoside solution (containing 6.6 mg/ml of lipopolysaccharide) and 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH 7.4), and the resulting mixture was stirred in a Vortex® mixer until uniform dispersion was achieved. Subsequently, 0.3 ml of anti-human T4 serum (5 mg Ab/ml) was added and stirred. The resulting mixture was placed in a dialyzing tube and dialyzed against 0.01 M HEPES buffer (pH 7.4) for 1 hour, after which the liposome suspension thus obtained was purified by ultra-centrifugation (35,000 r.p.m., 40 mm x 5). The pellets thus obtained were suspended in 5 ml of 0.01 M HEPES buffer (pH 7.4) to obtain encapsulated anti-T4 antibody. Example 1 Measurement of human T4 [Specimens] Mixtures of 50 µℓ of a 0.1% bovine serum albumin solution containing a predetermined concentration of human T4 and 150 µℓ of a pretreating agent (a 0.16 N NaOH solution containing 2.7% polyoxyethylene (10) octylphenyl ether). [Liquid reagents] Reagent 1 (R1): A solution of T4-labeled liposomes (containing glucose-6-phosphate dehydrogenase) 11 µmol/ml (in terms of cholesterol) the encapsulated anti-T4 antibody obtained in Referential Example 1 2 µmol/ml (in terms of cholesterol) oxidized form nicotinamide adenine dinucleotide (NAD) 3.2 mg/ml in 50 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer (pH 7.4) Reagent 2 (R2): A solution of a complement (guinea pig fresh serum) 0.25 ml/ml glucose-6-phosphate (G-6-P) 6.5 mg/ml [Procedure] To 20 µℓ of each specimen was added 200 µℓ of R1 and the resulting mixture was incubated at 37°C for 5 minutes. Then, 100 µℓ of R2 was added, and after further incubation at 37°C for 5 minutes, the change of absorbance at 340 nm was measured. [Results] The calibration curve thus obtained is shown in Fig. 1. It was prepared by plating absorbance (OD 340 nm ) on the axis of ordinate corresponding to individual T4 concentrations (µg/dl) on the axis of abscissa. As is clear from Fig. 1, a satisfactory calibration curve can be obtained by the immunoassay method based on complement-dependent immune lysis of microcapsules of this invention which uses the reagent combination for immunoassay method based on complement-dependent immune lysis of microcapsules of this invention. T4 was measured by use of the same reagent as described above by the same procedure as described above, except for using as specimens, mixtures of 50 µℓ of each of control sera I, II and III of Bio-Rad Laboratories and 150 µℓ of the aforesaid pretreating agent. The amount of T4 in each control serum was determined using the calibration curve obtained in the above. The results obtained are shown in Table 1. Control serum Measured value (µg/dl) Values indicated by Bio-Rad Laboratories (µg/dl) I 6.0 3.4 - 5.2 II 10.4 7.7 - 10.1 III 14.8 13.2 - 16.8 As is clear from the results shown in Table 1, satisfactory measurement results can be obtained by the process of this invention. Example 2 Stability test on reagent liquids [Specimens] Mixtures of 50 µℓ of a 0.1% bovine serum albumin solution containing a T4 concentration of 0, 10 or 25 µg/dl and 150 µℓ of the same pretreating solution as in Example 1. [Liquid reagent] R1: a liquid reagent prepared in the same manner as in Example 1 and stored at 15°C for a predetermined number of days. R2: the same as in Example 1. [Procedure] To 20 µℓ of each specimen was added 600 µℓ of R1 and the resulting mixture was incubated at 37°C for 5 minutes. Then, 300 µℓ of R2 was added, and after further incubation at 37°C for 5 minutes, the change of absorbance at 340 nm was measured. [Results] The measurement results are shown in Table 2. T4 concentration (µg/dl) Number of storage days (15°C) Immediately after preparation 2nd day 5th day Measured value (OD 340nm ) 0 0.722 0.700 0.700 10 0.455 0.450 0.440 25 0.221 0.215 0.217 As is clear from the results shown in Table 2, the reagents of this invention have a good stability. When T4 was measured by using as R1 a liquid reagent prepared by use of a conventional antibody solution in place of the encapsulated antibody of this invention, there was no difference among measured values (OD 340nm ) obtained for the specimens in the case where the T4 concentration was 0, 10 or 25 µg/dl (data not shown). Thus, it was clear that this liquid regent was of no practical use. Example 3 Measurement of human T4 in serum [Specimens] Mixtures of 50 µℓ of each of fresh sera of 25 human beings and 150 µℓ of a pretreating agent (a 0.16 N NaOH solution containing 2.7% polyoxyethylene (10) octylphenyl ether). [Liquid reagents] R1: the same as in Example 1. R2: the same as in Example 1. [Procedure] The same as in Example 1. Comparative Example 1 Measurement of T4 in human serum T4 was measured for the same fresh sera of 25 human beings as used in Example 3, by using a commercially available reagent for measuring T4 (Amerlex T4, mfd. by Amersham International plc.). The measurement was carried out according to the standard procedure described in the instructions. In Fig. 2 is shown a correlational graph prepared on the basis of measured values obtained by Example 3 and Comparative Example 1. The results of statistical processing of the measured values obtained by Example 3 and Comparative Example 1 are shown below: Correlation coefficient γ = 0.9806 Regression line formula Y = 1.057X - 0.75 wherein Y: the measured value obtained by Example 3 X: the measured value obtained by comparative Example 1 As is clear from the above results, the measured values of T4 obtained by measuring process of this invention were in good correlation with those obtained by a conventional method. As described above, the present invention provides an improved immunoassay method based on complement-dependent immune lysis of microcapsules which uses liquid regents of novel forms. The measuring process of this invention is markedly effective in that it permits application to autoanalyzers which is difficult for conventional methods. Therefore, this invention contributes greatly to the art.

Claims: A liquid reagent used for an immunoassay method based on complement-dependent immune lysis of microcapsules comprising: (a) first microcapsules having an analyte to be measured immobilized on surfaces thereof and containing a substance capable of acting as a label therein, wherein said first microcapsules are capable of being lysed by complement once activated by an antigen-antibody complex formed on the said surfaces, and (b) second microcapsules having different capsule walls from said first microcapsules and encapsulating an antibody specific to said analyte, wherein the capsule walls of said second microcapsules are more easily lysed by a second substance having a cell membrane lysing activity than the capsule walls of said first microcapsules. A liquid reagent according to claim 1, wherein said second microcapsules are liposomes. A liquid reagent according to claim 2, wherein said liposomes are prepared by a surfactant removal method. An immunoassay reagent combination used for an immunoassay method based on complement-dependent immune lysis of microcapsules comprising: (a) a first liquid comprising: (i) first microcapsules having an analyte to be measured immobilized on surfaces thereof and containing a substance capable to act as a label therein, wherein said first microcapsules are capable of being lysed by complement once activated by an antigen-antibody complex formed on the said surfaces, and (ii) second microcapsules having different capsule walls from said first microcapsules and encapsulating an antibody specific to said analyte, wherein the capsule walls of said second microcapsules are more easily lysed by a second substance having a cell membrane lysing activity than the capsule walls of said first microcapsules. (b) a second liquid containing complement. An immunoassay reagent combination according to claim 4, wherein said second microcapsules are liposomes. An immunoassay reagent combination according to claim 5, wherein said liposomes are prepared by a surfactant removal method. An immunoassay method based on complement-dependent immune lysis of microcapsules comprising: (a) a first step of reacting a sample containing an analyte to be measured and a substance other than the complement to be used when carrying out the second step, said substance having a cell membrane lysing activity, with a first liquid comprising: (i) first microcapsules having an analyte immobilized on surfaces thereof and containing a substance capable to act as a label therein, wherein said first microcapsules are capable of being lysed by complement once activated by an antigen-antibody complex formed on the said surfaces, and (ii) second microcapsules having different capsule walls from said first microcapsules and encapsulating an antibody specific to said analyte, wherein the capsule walls of said second microcapsules are more easily lysed by the said substance having a cell membrane lysing activity than the capsule walls of said first microcapsules, wherein the said substance is present in a concentration sufficient to lyse said second microcapsules but not said first microcapsules. (b) a second step of reacting the resulting reaction solution with a second liquid containing complement, and (c) a third step of measuring the amount of the substance capable of acting as a label liberated by the second step. An immunoassay method according to claim 7, wherein said second microcapsules are liposomes. An immunoassay method according to claim 8, wherein said liposomes are prepared by a surfactant removal method. An immunoassay method according to claim 7, wherein the substance capable of acting as a label is an enzyme, a substance which can emit fluorescence, a dye, a luminescent substance or a spin marker. An immunoassay method according to claim 10, wherein the substance capable of acting as a label is an enzyme. An immunoassay method according to claim 10, wherein the enzyme is glucose-6-phosphate dehydrogenase.

Inventors: KIDA MASAAKI, KUBOTSU KAZUHISA, MATUURA SHUJI, KIDA, MASAAKI, KUBOTSU, KAZUHISA, MATUURA, SHUJI
Applicants: WAKO PURE CHEM IND LTD, WAKO PURE CHEMICAL INDUSTRIES LTD
Application year: 1990
Main classification: G01N33
IPC codes: G01N33