JPH02162260A - Reagent for immunoassay - Google Patents

Abstract

PURPOSE:To develop high complementary activity even by either one of antibody and antigen immobilizing method and to quantify the antigen or antibody in a specimen to be examined with high sensitivity by preparing a liposome using haptenized phospholipid as a constitutional component. CONSTITUTION:An antibody or antigen is immobilized on the surface of a liposome containing phospholipid, haptenized phospholipid and cholesterol as principal constitutional components and a hydrophilic marker substance is sealed in the liposome. At this time, the ratio of phospholipid and cholesterol being the principal constitutional components of the liposome is pref. about 1:1. Further, it is necessary that haptenized phospholipid is 0.005-0.1mol per 1mol of phospholipid. This reagent, a specimen containing the antigen or antibody and a complement are mixed in a buffer solution to bring about the bonding reaction of antigen-antibody and the complement. Whereupon, the marker substance is discharged from the liposome in proportion to reaction quantity. Next, determination is performed by an analytical method corresponding to the marker substance and, for example, by a preliminarily prepared calibration curve, the amount of the antibody or antigen in the specimen can be measured.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an immunoassay reagent, and more specifically, to an immunoassay reagent, which is capable of measuring a specific antigen or antibody present in a sample with a simple operation and high sensitivity. Regarding reagents for immunoanalysis.

[Conventional technology]

Immunoassay methods that utilize antigen-antibody reactions are extremely important in clinical diagnosis of various endocrine diseases, and various measurement methods are known. Among these, in recent years, liposomes encapsulating markers are prepared and sensitized with antigens or antibodies, and the sensitized liposomes are allowed to coexist with the specimen, so that the antigens or antibodies covalently bound to the liposomes and the antibodies in the specimen are used. Alternatively, the antigen-antibody complex is formed by reacting with the antigen, this complex is specifically destroyed, and the marker flowing out from the liposome is measured, while the same sensitized liposome as above is reacted with various known amounts of antigen or antibody. A method has been proposed in which antibodies or antigens are quantified by coexisting with each other and preparing a standard calibration curve in advance by measuring the outflow marker in the same manner as above, and comparing the measurement results of the specimen with the standard calibration curve. This method is attracting attention because it is easy to operate.

[Problem to be solved by the invention]

The sensitized liposomes used in the above method include antibodies or antigens on the surface of the liposomes, such as N-succinimidyl 3-(2-pyridylthio)propionate (SPDP).
, N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N-succinimidyl 4-
(p-maleimidophenyl)acetate (SMP^)
N-succinimidyl 4-(pmadreimidophenyl)
propionate (SMPP), N-(γ-maleimidobutyryloxy)succinimide (GMBS) and N-
(ε-maleimidocaproyloxy)succinimide (
It is immobilized using a trifunctional reagent such as 8MC3) as a crosslinking agent. The present inventor also oxidized the antibody with periodic acid under mild conditions, and then bound it to a liposome containing a compound having a hydroxyamino group, a hydrazino group, a ureido group, or an amino group to sensitize the antibody. Method for obtaining liposomes (hereinafter referred to as "periodic acid method")
He discovered this and filed a patent application first.

However, in the case of sensitized liposomes immobilized by other immobilization methods, such as carbodiimide methods, carbamylation methods, and methods using active esters such as N-hydroxysuccinimide palmitate, the Even though an antigen-antibody reaction occurred, complement could not be activated sufficiently, so sufficient marker release could not be obtained, and it could not be used in the above immunoassay method.

[Means to solve the problem]

Under these circumstances, the present inventor conducted intensive research and found that if antibodies or antigens are immobilized on liposomes containing hubtenated phospholipids, complement activity is improved, and that any immobilization method can improve complement activity. It was discovered that the obtained sensitized liposomes can also be used in the above immunoassay method, and the present invention was completed.

In other words, the present invention provides for immobilizing antibodies or antigens on the surface of liposomes whose main components are phospholipids, nobutenated phospholipids, and cholesterol, and encapsulating a hydrophilic labeling substance within the liposomes. The present invention provides an immunoassay reagent characterized by the following.

The liposome of the present invention is composed of conventionally commonly used phospholipids, cholesterol and haptenized phospholipids.

As the haptenated phospholipid, trinitrophenyl (T
NP)-Cing Mitsukabroyl (Cap) L-α-dipalmitoyl phosphatidylethanolamine (DPPB),
Dinitrophenyl (DNP) -DPPB, RoNP-
Cap-〇PPB, fluorescein 7-inch ocarbamyl (
Fl)-DPPB, azobenzene arsonate (8B^
)-tyrosyl (Tyr)-roPP[l, dansyl (DN
S)-DPPB, 4-dimethylamino-azobenzene-
4'-sulfonyl (0^0S)-DPPB, maleimidobenzoyl (MO)-roPPB, dithiopyridyl (D
TP)-DPPB and the like.

The ratio of phospholipid and cholesterol, which are the main components of the liposome of the present invention, is approximately 1:1. (molar ratio) is preferred. In addition, habtenized phospholipid is 0.005% per mole of phospholipid.
It is necessary that the amount is ~0.1 mol; if it is less than this, the activation of complement will be insufficient, and if it is added in excess of this, the complement activity will be too strong, and the impurities in the sample will cause liposome Since it will be destroyed, accurate measurements cannot be made.

The labeling substance encapsulated within the liposome must be hydrophilic and capable of being quantified when eluted outside the liposome. For example, such substances do not exhibit fluorescence due to self-quenching at high concentrations, but at low concentrations (10-3
Fluorescent compounds such as carboxyfluorescein, which emit very strong fluorescence at (M or less); Luminescent compounds such as luminol and luciferin, which emit light by oxidation reaction outside the liposome; have a specific absorption band in the visible or ultraviolet region Light-absorbing compounds (water-soluble dyes, etc.); Sugars such as glucose and sucrose that are decomposed by the action of oxidative enzymes, resulting in oxygen consumption or hydrogen peroxide production; Relatively large ionic compounds such as tetrapentylammonium; Nicotinamide azo Herring nucleotide (NAO)
Coenzymes such as ); radical compounds such as methyl viologen are desirable.

However, enzymes are not used as labeling substances in the present invention. These compounds are appropriately selected in consideration of factors such as detection method, sensitivity, and stability of liposomes.

The immunoassay reagent of the present invention is produced, for example, by the following method. First, phospholipids, haptenized phospholipids, cholesterol, and if necessary, compounds having a functional group capable of binding to antibodies or antigens, such as DPPB, hydrazine compounds, hydroxylamine compounds, and hydrazone compounds, are added, and liposomes are formed by vortexing. Prepare. Specifically, a solvent is added to the above mixture and reacted, and then the solvent is distilled off and dried by suction. Thereafter, an aqueous solution of a predetermined labeling substance is added to a flask with a thin film formed on the wall, the flask is tightly stoppered, and the flask is shaken to obtain a suspension of liposomes.

On the other hand, in the case of the periodic acid method, antibodies or antigens are modified by oxidation using periodic acid, etc. under mild conditions such that only the sugar chain hydroxyl groups in the antibody molecule are oxidized to formyl groups. Antibody. In the case of an active ester method such as N-hydroxysuccinimide palmitate (NH3P), the antigen or antibody and the active ester are incubated to form a modified antibody.

Finally, the immunoassay reagent of the present invention can be obtained by reacting the liposome with the modified antibody in a buffer.

Such a reagent is usually obtained as a microcapsule containing a labeling substance and having an antibody or antigen immobilized on its surface.

Quantification of antigens or antibodies in a specimen sample using the immunoassay reagent of the present invention is carried out, for example, as follows. first,
The reagent, a sample containing antigen or antibody, and complement are mixed in a suitable buffer (eg, gelatin-veronal buffer) to cause a binding reaction between antigen-antibody and complement. Then, a labeling substance is released from within the liposome in proportion to the amount of reaction.

Next, quantification is performed using an analysis method depending on the labeling substance (e.g., fluorescence analysis if the labeling substance is a fluorescent substance), and, for example, the amount of antibody or antigen in the sample is measured using a calibration curve prepared in advance. can do.

The complement used in the quantitative operation is not particularly limited, but guinea pig serum is usually used. However, rabbit, mouse, human, etc. serum may also be used.

〔Effect of the invention〕

As mentioned above, the reagent for immunoassay of the present invention, in which liposomes are prepared using hubtenated phospholipids as a constituent, exhibits high complement activity regardless of the antibody or antigen immobilization method, and is highly sensitive in the test sample. antigen or antibody can be quantified.

〔Example〕

Next, an example will be given and explained.

Example 1 (i) Preparation of marker-encapsulated liposomes: oral PPC (10
mM, 100μl), cholesterol (10mM, 10
0μm), DTP-DPPB (1mM, ■No, ■5μ11■10μl or ■■20μ of the chloroform solution is placed in a 10al! flask, and the solvent chloroform is evaporated with an evaporator. A film-like substance will adhere to the inner surface of the flask. After vacuum-drying this film for 1 to 2 hours, 100 μl of a 0.IN Na0tl solution of carboxyfluorescein used as a marker was added, and the film was dried for 5 to 10 minutes using a portex mixer.
Stir vigorously for a minute.

Then, excess carboxyfluorescein was added to 10,000
By centrifuging at XG, a liposome (MLV type) in which carboxyfluorescein is encapsulated as a marker is obtained.

The liposomes were prepared in 0.01 M HBPBS buffer (0.0
, 15M, NaCl5pH 7,45) 500p
Suspend in 1 and store.

(ii) Modified antibody preparation! il: 30a+M NH3P in dimethylformamide solution lO
μl 2% octyl glucoside (surfactant).

1 ml). In addition to this, goat I@G (2 mg/m
ff1. Add 1 ml and incubate at 37°C for 16 hours. This is gel-filtered with Sephadex G-50 to remove the surfactant, and a protein fraction (1 mg/-) is collected and used as a modified antibody.

(iii) Antibody sensitization of liposomes: Add 500 μl of the modified antibody solution (1/d) obtained in (ii) to 500 μl of the liposome suspension obtained in (i), and incubate at 4°C for 16 to 30 minutes.
The mixture was incubated for 20 hours and centrifuged to obtain an antibody-sensitized liposome, which is an immunoassay reagent of the present invention.

The antibody-sensitized liposomes obtained in this way are gelatin-
Suspend in veronal buffer and store at 4°C.

(iv) Release assay with rabbit anti-goat IgG
: For dilution, gelatin-veronal buffer (GVB-)
with 0.1 mM MgC1* and 0.03 mM [:
A buffer solution (GVB''a) containing aCli was used. For the measurement, the antibody-sensitized liposome storage solution prepared in (iii) was diluted 300 times with GVB''+.

In addition, guinea pig complement was similarly diluted with GVB"+ to a volume of 1 m5 units. Measurements were carried out as follows. In a 96-well microplate, the sample [rabbit anti-goat Ig
Dispense 325 μl of G (X 10” to ×10’) and then add 25 μl of the 300-fold diluted liposome suspension.
1 and 25 μl of complement (3 units). 37℃.

After reacting for 1 hour, add 10
Add 100 μl of veronal buffer containing +mM HOT^. Since the amount of antigen in the sample is proportional to the amount of carboxyfluorescein released from the liposome,
The amount of fluorescence emission of 530nI11 is measured.

Incidentally, the fluorescence intensity was expressed as marker release % with respect to the amount of fluorescence obtained by destroying liposomes with a surfactant as 100%.

The results are shown in FIG. In Figure 1, the curve ■ is DTP-
No DPPB added, curve ■ is 5μ! Addition, curve ■ is 10μ
l addition, curve ■ is 20μ! Additions are shown. As shown in FIG. 1, it can be seen that complement activity was improved by the addition of DTP-DPPH.

Example 2 (i) Preparation of marker-encapsulated liposomes: DPPC:Cholesterol: []PPB:DTP-[]PPEI in 1 part
: 1 : 0.1: (0,0,005,0,01
, 0, 02) to obtain liposomes in the same manner as in Example 1 (i).

(ii) Preparation of modified antibody: Anti-CRP antibody (goat IgG fraction) (4,6■/-1
500 μl) in 0.1 MI acid buffer (pH 4,0
) Buffer exchange was performed by gel filtration on Sephadex G25. Obtained protein fraction 1.6
ml (4.3 mg protein/af), and add 0.0 mL of sodium periodate to it. In addition, 2
It was left in a water bath at 5°C for 30 minutes. The reaction was then diluted with 0.15M NaCl. Monthly carbonated buff y
- (pH 9,2) and gel filtration with Sephadex G25, protein fraction 1 - (approximately 1.9 mg protein/rn
l) was obtained.

(iii) Antibody sensitization of liposomes: The liposome pellet obtained in (i) was added to the protein fraction (modified antibody) obtained in (11), and mixed by inversion at 4°C overnight. Then,
Centrifugal washing with GVB-, 1 ml GVB- (0,1% Na
A goat IgG-conjugated liposome (hereinafter abbreviated as "anti-CRP liposome") was obtained by suspending it in Na-containing solution.

(iv)' Quantification of CRP: In a 96-well microplate, add 25μ11 of anti-CRP liposomes diluted 400 times with 6V Ro◆3 and 25 μl of purified CRP antigen.
Add μl and incubate for 1 hour at 37°C. 25μ of anti-CRP antibody (rabbit serum) diluted 100 times with this
11 complement <4C11,. ) was added and incubated at 37°C for 1 hour. Thereafter, the complement reaction was stopped in the same manner as in (iv) of Example 1, and the amount of fluorescence emitted was measured to determine the marker release %.

The results are shown in FIG. In Figure 2, the curve ■ is the mouth TP-
2) No addition of PP[l, curve 2 indicates addition of 0.005, curve O indicates addition of Q, (addition of In, curve O indicates addition of 0.02.

Example 3 1) PPI3 for (i) to (iii) of Example 2
Anti-CRP II posomes were obtained in the same manner except that ε-Cap-OPP[l was used instead of ε-Cap-OPP[l. Using this, the same procedure as in Example 2 (iv) was performed to determine the marker release %.

The results are shown in FIG. In Figure 3, the curve ■ is 0TP-
2) No addition of PP8, curve ■ indicates addition of 0.01, curve ■ indicates 0.01 addition.
02 addition is shown.

[Brief explanation of the drawing]

Figure 1 shows DTP-〇PPB when rabbit anti-goat IgG antibody was measured using the immunoassay reagent obtained in Example 1.
FIG. 3 is a diagram showing the relationship between the amount added and marker release. FIG. 2 is a diagram showing the relationship between the amount of DTP-DPPB added and marker release when CRP antigen was measured using the immunoassay reagent obtained in Example 2. Figure 3 shows Example 3
FIG. 3 is a diagram showing the relationship between the amount of DTP-DPP [l added and marker release when CRP antigen was measured using the immunoassay reagent obtained in FIG. that's all

Claims (1)

[Scope of Claims] 1. Immunization characterized by immobilizing antibodies or antigens on the surface of liposomes whose main components are phospholipids, haptenized phospholipids, and cholesterol, and encapsulating a hydrophilic labeling substance within the liposomes. Analytical reagents. 2 Haptenated phospholipid is 0.0% per mole of phospholipid.
2. The reagent for immunoassay according to claim 1, wherein the amount is 0.005 to 0.1 mol.