JPH0799373B2 - Antigen quantification method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for quantifying an antigen, and in particular, a marker-encapsulated liposome is sensitized with an antibody by a covalent bond, and the antibody-sensitized liposome and a free antibody are used as a sample. To form a sandwich type antigen-antibody complex with the antigen in the sample in coexistence with the antibody, the antibody covalently bound to the liposome, and the free antibody,
A method for quantifying an antigen in which a marker flowing out from the liposome is measured by activating complement with this complex and specifically destroying the liposome, and the measured value is collated with a separately prepared standard calibration curve. .

Therefore, the present invention can be used for the purpose of diagnosing various endocrine diseases, infectious diseases, tumors and the like.

(Prior art and its problems) Immunoassays utilizing antigen-antibody reactions have become indispensable for clinical diagnosis of various endocrine diseases, infectious diseases, tumors and the like. This immunoassay can be roughly classified into a labeling method and an unlabeling method.

Among these, various labeling substances (markers) such as radioisotopes, fluorescent substances, enzymes or enzyme-related substances have been used to carry out a labeled immunoassay having excellent sensitivity. Although radioisotopes have been conventionally used as markers in terms of sensitivity, radioisotope reagents are unstable due to radioisotopes having a half-life, and facilities and management costs for preventing radiation damage are high. Further, there is a problem in that it becomes radioactive waste even after use. Therefore, the measuring method using a fluorescent substance or an enzyme as a marker is becoming mainstream together with the research on the increase in its sensitivity.

Immunoassays that use a fluorescent substance or enzyme as a marker currently require a step of separating (B / F separation) those bound to the antigen or antibody to be measured and those not bound in the marker. There is a method using a heterogeneous system and a method using a homogeneous system which does not require such a separation step.

Immunoassays that use a heterogeneous system include the two-antibody method and the solid-phase method, which are also used in radioisotope-labeled immunoassays, but these methods separate unreacted and reacted substances. However, there is a problem that it is difficult to carry out this separation step, and thus it is difficult to speed up the quantification. On the other hand, in an immunoassay method using a homogeneous system, when quantifying the antigen, the marker-encapsulated liposome is sensitized with the antigen, and the antigen-sensitized liposome and the antigen in the sample are used in the reagent. Antigen-sensitized liposomes and the above free antibodies are allowed to compete with each other in the presence of the free antibody to form an antigen-antibody complex, and the liposome in which this complex is formed is specifically destroyed by complement and effluxed. This is usually done by measuring a marker that In this case, the amount of antigen in the sample is inversely proportional to the amount of marker outflow. This general method has problems in that unstable antigens and difficult-to-obtain antigens cannot be measured because the antigens are sensitized to liposomes and that the measurement range is narrow.

Recently, of two kinds of antibodies of different immunized animal species, one of the antibodies is allowed to react with the liposome, and the other antibody and complement are further added to destroy the liposome. It has been proposed to release the substance contained in the liposome and measure the substance to quantify the antigen (JP-A-60-138465). In this method, goat and rabbit are mainly used as immunized animals in the case of polyclonal, but goat-derived antibody does not activate complement (mainly guinea pig complement),
On the other hand, a rabbit-derived antibody causes a nonspecific reaction when bound to the liposome surface, and therefore, a goat antibody is used as the antibody bound to the liposome and a rabbit antibody is used as the free antibody. According to this method, there is a proportional relationship between the amount of antigen in the sample and the amount of marker outflow.
Since an antibody obtained by immunizing two kinds of animals with one antigen is used, it is troublesome to prepare the antibody, and it is difficult to construct a stable measurement system that can measure the antigen with good sensitivity even in a low concentration range. There is.

(Object of the Invention) Therefore, an object of the present invention is to provide an antigen quantification method which has high measurement sensitivity, a wide measurement range, and is stable.

(Means and Actions for Solving the Problem and Achieving the Object) According to the present invention, the above-mentioned problem in the prior art is to sensitize a marker-encapsulated liposome with an antibody by a covalent bond. Coexist with the specimen to form a sandwich-type antigen-antibody complex with the antigen in the specimen, the antibody covalently bound to the liposome, and the free antibody, and activate the complement with this complex. A marker that flows out from the liposome is measured by specifically destroying the liposome, and the measured value is compared with a separately prepared standard calibration curve. Rabbit lgG is used as a free antibody, or a mouse monoclonal antibody is used as an antibody for sensitizing liposomes and the above-mentioned monoclonal antibody is used as a free antibody. The above-mentioned object is achieved as well as being solved by a method for quantifying an antigen, which is characterized in that a mouse monoclonal antibody or a rabbit lgG having a different recognition site for antigen determination is used as a clonal antibody.

As the complement used in the quantification method according to the present invention, animal species can be appropriately used, but since the titer is high, the dilution ratio can be remarkably increased (100-200 times), and therefore it is economical. Preferably, guinea pig complement is used.

Although the liposomes specifically prepared in Examples and the like described later are large liposomes (MLV) having multiple membranes, even large unilamellar liposomes (LUV) are small liposomes (SUV). But it doesn't matter. As the marker enclosed in the liposome, any substance commonly used in the present technical field such as a fluorescent substance, an enzyme, and an enzyme-related substance can be adopted. Fab 'is used when the antibody sensitized to the liposome is an antibody obtained by immunizing a rabbit, but when a rabbit-derived antibody is bound to the liposome, a nonspecific reaction is caused. The causative part is Fc, so F (ab ') ₂ can also be used. The antigens and antibodies specifically shown in the examples are exclusively derived from C-reactive protein (CRP), but they can be changed to any ones. N-Hydroxysuccinimidyl-3- (2-pyridyldithio) propinate (SPDP) was also used as a reagent for sensitizing liposomes with an antibody, but it may be any other bifunctional reagent, for example, It can be changed to N, N'-o-phenylenedimaleimide, N-succinimidyl-p- (N-maleimidophenyl) -4-butyrate, N-sulfosuccinimidyl-m- (N-maleimido) benzoate and the like. .

(Effect of the Invention) The method of the present invention comprises sensitizing a marker-encapsulated liposome with an antibody,
The antibody-sensitized liposome and the free antibody are allowed to coexist with a sample to form a sandwich-type antigen-antibody complex with the antigen in the sample, the antibody bound to the liposome, and the free antibody. It is a method for quantifying antigens in which the marker flowing out from the liposome is measured by activating complement to specifically destroy the liposome, and the measured value is compared with a separately prepared standard calibration curve. Rabbit Fab 'is used as the antibody for sensitization and rabbit lgG is used as the free antibody, or mouse monoclonal antibody is used as the antibody for sensitizing liposomes, and the antigen-recognition recognition site is different from the above monoclonal antibody. Mouse monoclonal antibodies or rabbit lgG are used.

As a result, the following advantages are brought about.

(1) Since it is a homogeneous measurement system, a washing operation for separating already reacted substances and unreacted substances, so-called B / F separation, is unnecessary, and therefore the operation is easy.

(2) Since the antigen is sandwiched by two kinds of antibodies, a relatively unstable antigen can be used as a measurement target and the quantitative determination of an antigen having a risk of infection such as a viral antigen can be performed safely. You can

(3) When the complement is activated by the sandwich type antigen-antibody complex to destroy the marker-encapsulated liposome, the outflow rate of the marker increases in proportion to the amount of the antigen in the sample, so that the measurement accuracy is high and the measurement is high. The range is wide.

(4) The preparation is easy when the antibody bound to the liposome and the free antibody have the same animal origin.

(5) If two types of mouse monoclonal antibodies having different antigenic recognition sites are used as the antibody bound to the liposome and the free antibody, the specificity can be improved and the measurement sensitivity can be increased. Further improvement in sensitivity can be expected by appropriately selecting the type.

(Examples, etc.) Next, the present invention will be described in more detail and specifically with reference to Reference Examples and Examples.

The reagents and the like used in Reference Examples and Examples were prepared as follows.

Serum Fresh guinea pig serum was used as a complement source, and human serum derived from healthy volunteers was used as CRP-free human serum and stored at −75 ° C. until use.

One unit of complement titer (1U, 1CH ₅₀ ) is 60 at 37 ℃.
Sheep red blood cells (SRB
C, 5 × 10 ⁸ /7.5 ml) is defined as the amount of guinea pig serum required to hemolyze 50%.

Purification of CRP antigen 50 μg / ml of CRP antigen derived from patients with inflammatory diseases
The pooled serum contained above was filtered through a 0.45 μm Millipore filter and purified by affinity chromatography using an agarose gel bound with a 2-aminoethanol dihydrogen phosphate ligand. The prepared CRP antigen showed a single protein by electrophoretic analysis using 7.5% SDS-polyacrylamide gel. CRP concentration is SRI
It was measured by the D method (“N-lmmun manufactured by Nissui Pharmaceutical Co., Ltd.
oring CRP "). The purified CRP was stored at -75 ° C, and each standard concentration solution was prepared by appropriately diluting gelatin-veronal buffered saline (GVB, pH 7.4) at the time of use.

Preparation of Anti-CRP Polyclonal Antibody (Pr) Antiserum against human CRP was induced by immunizing rabbits with an emulsion of purified CRP solution and Freund's complete adjuvant (Difco Lab.). Monospecific antibody was obtained by passage through a CRP negative human serum conjugated Sepharose 4B column. lgG is 0.05M Tris
-DE-52 cellulose (Whatm
The antiserum was fractionated by passing through a packed column (manufactured by an) and then precipitating with 33% ammonium sulfate. The lgG fraction was then passed through a CRP-loaded Spharose 4B column with glycine buffer (pH 2.5).

This antibody is used as a free antibody.

Preparation of Fab ′ fraction Fg was obtained by digesting the above purified lgG with pepsin at pH 4.5.
An (ab ') ₂ dimer was prepared. This dimer fragment was treated with mercaptoethyleneamine at pH 6.5 to give Fab 'monomer fragment.

This antibody is used for sensitizing liposomes, but the above-mentioned dimer can also be used.

Preparation of marker-encapsulated liposomes Each lipid [DPPC (dipalmitoylphosphatidylcholine, 0.5 μmol): Chol (cholesterol, 0.5 μm) was placed in a 10 ml flask.
mol): DTP-DPPE (dipalmitoylphosphatidylethanolamine with dithiopyridyl introduced by an amide bond, 0.01-0.05 μmol) is added and dissolved in chloroform. The lipid solution was treated in a rotary evaporator at 37 ° C. to volatilize the solvent chloroform to form a film on the inner wall of the flask, and the film was vacuum dried for another hour. Next, 100 ml of 0.1N NaOH solution of carboxyfluorescein (CF) as a marker
And stir vigorously for 10 minutes on a vortex mixer.

After that, the excess CF is removed by centrifugation at 10,000 × g to obtain the desired CF-encapsulated liposome (MLV type).

This liposome is 0.01M HEPES buffered saline (pH7.4)
Suspend in 500 μl and save.

Preparation of Polyclonal Antibody-sensitized Liposomes 500 μl (total lipid amount 1 μmol / ml) suspension of the above-described marker-encapsulated liposomes (lipid composition of liposome, DPPE: Chol: DTP-DPPE = 1: 1: 0.2) was prepared. Add to 500 μl of anti-CRP rabbit Fab ′ fraction as described above and incubate at room temperature overnight with gentle agitation. Unbound Fab 'is removed by repeating centrifugation (20000 xg) in GVB for 20 minutes at 4 ° C. After the final centrifugation, the pellet is suspended in 1 ml GVB and stored at 4-6 ° C. The sensitized amount of lgG to the liposome is estimated from the difference in protein concentration in the supernatant before and after the coupling reaction.

Preparation of anti-CRP monoclonal antibody a) Monoclonal antibody I (M ₁ ) Hybridomas producing antibodies against human CRP antigen were splenocytes and myeloma cells (P3-X63-Ag8) derived from BALB / c mice immunized with CRP antigen. -Ul). That is, purified CRP antigen (30 μg /
) And Freund's complete adjuvant (manufactured by Difco Lab.)
An intraperitoneal administration of an emulsion consisting of
After 30 days, 10 μg of CRP antigen was further injected intravenously for secondary immunization, and after 3 days, the splenocytes for fusion were obtained by slaughter, and the splenocytes were fused with the above-mentioned myeloma cells.

Hybridomas that specifically reacted with the CRP antigen were screened by the LILA method and cloned by the limiting dilution method.

b) Monoclonal antigen II (M ₂ ) This antibody was prepared using ascites fluid of mice immunized by intraperitoneal administration of the above hybridoma suspension. That is, ascites was collected and treated with 50% ammonium sulfate solution, and the antibody fraction was further purified by a DE-52 cellulose column using 0.05M Tris-hydrochloric acid buffer (pH8.0) containing 0.1M MaCl. Obtained.

Preparation of anti-CRP monoclonal antibody-sensitized liposome The procedure is the same as in the case of sensitizing the above-mentioned polyclonal antibody except that the above monoclonal antibody is used.

Reference Example As described in the section “Preparation of Polyclonal Antibody-sensitized Liposomes” above, except that rabbit Fab ′ at various concentrations was sensitized as an antibody (Fab ′ is mediated by its thiol residue, DTP- Binding to DPPE-containing liposomes), the relationship between the concentration of antibody used for sensitization and the amount of sensitization was investigated.

The results are shown in FIG. 1, and it was found that the concentration of the antibody used for sensitization and the sensitization amount are in a proportional relationship.

Example 1 1 μmol of DPPC anti-CRP rabbit Fab 'was added to the marker-encapsulated liposome described in the above section "Preparation of marker-encapsulated liposome".
5 μl of a 100-fold dilution of a liposome suspension sensitized to 280 μmol and a standard CRP antigen solution with a known CRP value, which was appropriately diluted as a sample (see the above-mentioned “Purification of CRP antigen”). ) 25 μl, and a CRP antibody in which the CRP antigen in the sample was sensitized to the above liposomes and a free antibody (the rabbit lgG fraction described in the above-mentioned “anti-CRP polyclonal antibody”, concentration 7 mg / ml, 25 μl) to form an antigen-antibody complex, and then 25 μl of guinea pig complement with a titer of 2CH ₅₀ was added and incubated at 37 ° C. for 1 hour. Then, 100 μl of veronal buffered saline (EDTA-VBS, pH 7.4) containing 10 mM disodium ethylenediaminetetraacetate was added to terminate the complement reaction.

The relationship between the amount of CRP antigen in the sample and the amount of released marker was as shown in FIG.

From this figure, it is found that the release rate of the marker increases in proportion to the amount of the antigen in the sample, and therefore the antigen amount can be measured for the sample of which the antigen amount is unknown by measuring the release rate of the marker.

In addition, although the measurement range of the antigen amount is shown only up to 300 ng / ml in FIG. 2, it can be sufficiently quantified up to about 500 ng / ml.

Example 2 1) Preparation of marker-encapsulated liposomes In the section "Preparation of marker-encapsulated liposomes" described above, except that DPPC, Chol, and DTP-DPPE were used as lipids at a molar ratio of 1: 1: 0.4. Marker-encapsulated liposomes were obtained by processing as described.

2) Dithiopyridylation of antibody Anti-CRP antibody dialyzed with 0.01 M HEPES buffer [The monoclonal antibody 1 (for test, M ₁ ) or "anti-CRP described in the above section" Preparation of anti-CRP monoclonal antibody "] [Preparation of Polyclonal Antibody] Goat polyclonal antibody prepared by operating in the same manner as in the case of the rabbit polyclonal antibody described in the section of “Preparation of polyclonal antibody (for control, Pg)”]
Add ethanol solution of SPDP to 00μM, react at room temperature for 30 minutes, then 0.1M acetate buffer (pH 4.5)
Gel filtration on a Sephadex G-25 column equilibrated with to give the desired dithiopyridylated antibody.

3) Thiolation of antibody Add 50 mM to the dithiopyridylated antibody solution obtained in 2) above.
Dithiothreitol was added to the reaction mixture at room temperature for 30 minutes, and then gel filtration was performed on a Sephadex G-25 column equilibrated with 0.1 M HEPES buffer (pH 7.5) to give the desired thiolated antibody. can get.

4) Antibody sensitization of liposomes The thiolated antibody solution obtained in the above 3) is added to the above 1)
Add the marker-encapsulated liposome suspension obtained in the above section, and
The desired antibody-sensitized liposomes can be obtained by gently stirring for a time and then centrifuging to remove unbound antibody.

5) Quantification of CRP antigen 5 μl of antibody-sensitized liposomes obtained in the above 4), 25 μl of free antibody, and various standard CRP antigen solutions which are samples with known CRP values (the above-mentioned “purification of CRP antigen”). 25 μ)
CRP in the sample was sandwiched between the antibody sensitized to liposomes and the free antibody to form an antigen-antibody complex, and then 25 μl of guinea pig complement with a titer of 2CH _50.
Was added and incubated at 37 ° C. for 1 hour. afterwards,
To terminate the reaction, 100 μl of veronal buffered saline (EDTA-VBS, pH 7.4) containing 10 mM disodium ethylenediamine tetraacetate was added.

As the free antibody, anti-CRP mouse monoclonal antibody II (having an antigenic recognition site different from that of monoclonal antibody I) described in the above-mentioned “Preparation of anti-CRP monoclonal antibody”, A mouse monoclonal antibody (monoclonal antibody III, M ₃ ) that has the same antigenic recognition site as anti-CRP rabbit polyclonal antibody or mouse monoclonal antibody II described in "Preparation of anti-CRP polyclonal antibody" but different in sensitivity is used. Was done.

A first antibody for sensitizing liposomes in this example,
The combination with the second antibody, which is a free antibody, is as follows. 1st antibody 2nd antibody M ₁ M ₂ M ₁ Pr Pg Pr (combination of controls) M ₁ M ₃ Guinea pig Release rate of marker that specifically destroys liposomes by action of complement and CRP antigen in specimen The results of examining the relationship with the amount were as shown in FIGS. 3 and 4. As a result, when a goat polyclonal antibody is used as the first antibody and a rabbit polyclonal antibody is used as the second antibody, the measurement sensitivity in the low concentration range is low, and when a mouse monoclonal antibody is used as the first antibody and a rabbit polyclonal antibody is used as the second antibody. Although it is possible to improve the measurement sensitivity in the low concentration range, a decrease in the marker release rate in the high concentration range, that is, a so-called "hook phenomenon" is slightly observed, and there are two types of antibodies having different antigen-determining recognition sites as the first and second antibodies. By using the mouse monoclonal antibody described above, the hook phenomenon can be suppressed, a highly sensitive measurement system can be constructed, and the sensitivity can be further improved by appropriately selecting the type of the monoclonal antibody. There was found.

[Brief description of drawings]

FIG. 1 shows that the constituent lipids are DPPC: Chol: DTP-DPPE = 1: 1: x, and liposomes having different DTP-DPPE molar ratios were prepared.
Each of these liposomes was sensitized with various concentrations of rabbit Fab 'antibodies, and the relationship between the antibody concentration used for sensitization and the amount of sensitization was examined. The Fab 'antibody was sensitized and the rabbit polyclonal antibody (lgG fraction) was used as a free antibody to sandwich the antigen in the sample to form an antigen-antibody complex, and this complex activated complement to destroy the liposome. A graph showing the results of investigating the relationship between the amount of antigen in the sample and the release rate of the marker,
Fig. 3 is the same as Fig. 2, except that the combination of antibodies is mouse monoclonal antibody (M ₁ ) -mouse monoclonal antibody (M ₂ ), mouse monoclonal antibody (M ₁ ) -rabbit polyclonal antibody (Pr) (both are tested. Combination) or goat polyclonal antibody (Pg) -rabbit polyclonal antibody (Pr) (control combination), a graph showing the results of examining the relationship between the amount of antigen in the sample and the release rate of the marker, and FIG. Similar to FIG. 3, but the antigen in the specimen when the combination of antibodies is mouse monoclonal antibody (M ₁ ) -mouse monoclonal antibody (M ₂ ) or mouse monoclonal antibody (M ₁ ) -mouse monoclonal antibody (M ₃ ). It is a graph which shows the result of having investigated the relationship between the amount and the release rate of the marker.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 21/00 Z 9282-4B

Claims (2)

[Claims] 1. A marker-encapsulated liposome is covalently sensitized with an antibody, and the antibody-sensitized liposome and a free antibody are allowed to coexist with a specimen, and the antigen in the specimen and the antibody covalently bound to the liposome are free. A sandwich-type antigen-antibody complex is formed with different antibodies, and the complex is activated to specifically destroy the liposome, thereby measuring the marker flowing out from the liposome. In the method for quantifying an antigen to be compared with the prepared standard calibration curve, rabbit Fab ′ is used as an antibody for sensitizing liposomes and rabbit lgG is used as a free antibody, or a mouse monoclonal antibody is used as an antibody for sensitizing liposomes and As a free antibody, a mouse monoclonal antibody or a rabbit having a different antigenic recognition site from the above-mentioned monoclonal antibody A method for quantifying an antigen, characterized in that GilgG is used. 2. The method for quantifying an antigen according to claim 1, wherein the complement is guinea pig complement.