JP2668403B2 - ATL antibody measurement reagent - Google Patents

Description

The present invention relates to a reagent for measuring an adult T-cell leukemia (ATL) antibody, and more particularly, to a simple automated method using a complement-dependent liposome membrane damage reaction. The present invention relates to an ATL antibody measurement reagent capable of measuring an ATL antibody by a manual operation.

[Conventional technology and its problems]

Numerous studies have been conducted on ATL occurring frequently in the southwestern part of Japan, other than that reported by Takatsuki et al. In 1977. According to Hinuma et al. In 1981, ATL patient-derived cells (MT-1)
Of HTLV-1 from the virus-infected cells (MT-1,
The antibody in the serum of the ATL patient was illuminated by the indirect fluorescent antibody method using MT-2) as an antigen, and the antibody-positive person was illuminated to be a virus carrier.

At present, blood transfusions containing blood cells and the high rate of viral carrier accumulation in families have revealed that star infection and marital transmission via lymphocytes in breast milk are the main natural transmission routes. It is said that the control of E. coli is the most effective in preventing infection. Therefore, the understanding of HTLV-1 carriers should not only be aimed at the differentiation of the virus-associated special disease type in leukemia patients, but should also be incorporated into general examinations for blood donors and pregnant women at the time of screening and hospitalization. It is high.

Conventionally, the indirect fluorescent antibody method, E
Although the IA method, the agglutination method, the Western-blot method, and the like have been adopted, automation of the measurement method is desired in view of the clinical importance of HTLV-1 as described above. However, the indirect fluorescent antibody method and the Western-blot method are complicated in operation, and are difficult to automate. Since the EIA method requires a washing operation,
There is a problem that a cleaning mechanism is required for automation. In addition, the agglutination method has a problem that although the operation is relatively simple, since the judgment is visual, individual differences are caused in the measurement results.

[Means for solving the problem]

Under such circumstances, the present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the above objects can be achieved by utilizing a complement-dependent liposome membrane damage reaction, and have achieved the present invention. completed.

That is, the present invention binds an ATLV-related antigen solubilized with sodium deoxycholate through a cross-linking agent to the surface of a liposome containing phospholipids and cholesterol as main constituents, and hydrophilically labels the inside of the liposome. An object of the present invention is to provide an ATL antibody measurement test characterized by enclosing a substance.

In the present invention, the liposome may be any of those conventionally used as long as the liposome contains phospholipid and cholesterol as main components, but is stable when the ratio of phospholipid to cholesterol is about 1: 1. Liposomes are easy to obtain. Further, the fatty acid residue in the phospholipid preferably has 12 to 18 carbon atoms, and more preferably has an even number.

The labeling substance encapsulated in the liposome must be hydrophilic and can be quantified when eluted out of the liposome. Such a substance, for example, does not show fluorescence due to self-quenching at a high concentration, but has a low concentration (10 ⁻³ M
Fluorescent compounds such as carboxylfluorescein that emits extremely strong fluorescence in the following); issuing compounds such as luminol and luciferin that emit light by an oxidation reaction outside the liposomes; absorption that has a specific absorption band in the visible or ultraviolet region. Compounds (water-soluble dyes, etc.); sugars such as glucose and sucrose that are decomposed by the action of oxidase and cause oxygen consumption or hydrogen peroxide production; relatively large ionic compounds such as tetrapentyl ammonium; nicotinamide adenine dinucleotide Coenzymes such as (NAD); radical compounds such as methylpiologen are desirable.

The ATLV-related antigen of the present invention includes ATL virus, MT-
Those obtained by solubilizing 2 etc. with sodium deoxycholate or the like are used.

The ATL antibody measuring reagent of the present invention is produced, for example, by the following method.

First, a phospholipid and cholesterol are placed in a flask, and a solvent is added to cause a reaction. Then, the solvent is distilled off, followed by suction drying. Thereafter, an aqueous solution of a predetermined labeling substance is added to the flask having the thin film formed on the wall surface, sealed, and shaken to obtain a labeling substance-encapsulated liposome.

On the other hand, the ATLV-related antigen is reacted with a crosslinking agent in a buffer to introduce a crosslinking group, and then, if necessary, further reacted with a reagent for reducing the crosslinking group (eg, dithiothreitol; DTT). To obtain a modified antigen.

Finally, the liposome encapsulating the ATLV-related antigen of the present invention can be obtained by reacting the labeled substance-encapsulated liposome with the modified antigen in a buffer. Such an ATL antigen measurement reagent is usually obtained as a microcapsule containing a labeling substance and carrying an antigen immobilized on the surface.

Examples of the crosslinking agent in the above production method include N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), N-succinimidyl 4- ( p-maleimidophenyl) acetate (SMP
A), N-succinimidyl 4- (p-maleimidophenyl) propionate (SMPP), N- (γ-maleimidobutyryloxy) succinimide (GMBS) and N- (ε
-Maleimidocaproyloxy) succinimide (EMC
S).

Using the ATL antibody measurement reagent prepared in this way,
To measure the ATL antibody in the subject, the reagent and complement are added to the subject to cause a binding reaction between the antigen-antibody and complement. Then, the labeling substance is released from the liposome in proportion to the reaction amount. Next, quantification is performed by an analysis method corresponding to the labeling substance (for example, if the labeling substance is a fluorescent substance, a fluorescence analysis method), and for example, the amount of the antibody in the sample is measured by a calibration curve prepared in advance. You can

The complement used in this measurement operation is not particularly limited, but guinea pig serum is usually used. But,
The formation of rabbits, mice, humans and the like may be used.

〔The invention's effect〕

As described above, by using the ATL antibody measurement reagent of the present invention, ATL antibodies in a subject can be accurately measured by a simple operation, and furthermore, the automation thereof is possible.

〔Example〕

 Hereinafter, the present invention will be further described with reference to examples.

Example 1 (1) Preparation of ATLV-related antigen: Purified ATL virus purchased from Cytotech was treated with sodium deoxycholate to obtain a solubilized protein. The solubilization treatment was performed by the following method. Add the ATL virus suspension (2 mg / ml) to 20 ml of veronal buffer (pH 7.5) containing 0.2% sodium deoxycholate, and add
For 20 hours. After stirring, the mixture was centrifuged, the supernatant was separated, and further concentrated. This is 0.1M NaCl containing 0.01M
After dialysis against Hepes buffer (pH 7.5), the solution was centrifuged to obtain a solubilized protein. In a similar manner, antigens obtained from MT-2 cells can also be solubilized.

(2) Preparation of ATLV antigen-sensitized liposome: (i) Preparation of liposome 1 μmol of dipalmitoylphosphatidylcholine (DPPC), 1 μmol of cholesterol (Chol), and dithiopyridylated dipalmitoylphosphatidylethanolamine (DTP-DPPE) ) 0.05 μmol was placed in a pear-shaped flask, and chloroform in which lipid was dissolved was distilled off by an evaporator. After drying in a vacuum desiccator for an additional hour, 0.2 μm carboxyfluorescein (CF)
Was shaken vigorously and the lipid thin film on the glass wall of the pear-shaped flask was peeled off to prepare CF-encapsulated liposomes.
Unencapsulated CF was prepared using 0.01M Pes buffer (containing 0.15M NaCl; pH
In 7.5), centrifugal washing was performed three times to separate.

(Ii) Sensitization of ATLV-related antigen to liposome ATLV-related protein (1.5 mg / ml) solubilized in (1) was centrifuged,
The supernatant was collected. 30 mM N-succinimidyl-3
-(2-Pyridylthio) propionate (SPDP) 2.0μ
Was added and reacted at room temperature for 30 minutes. Subsequently, in order to remove excess SPDP, gel filtration was carried out on Sephadex G-25 equilibrated with 20 mM MES buffer (pH 6.0). The protein fraction (1.5 ml) obtained from the gel filtration was added with 20 mg of dithiothreitol (DTT), reacted at room temperature for 30 minutes, and equilibrated in advance to 0.01 M with a Pes buffer. The mixture was subjected to gel filtration with Sephadex G-25 to separate an excess of DTT and a protein fraction. Protein fraction 1m thus obtained
The liposome pellet obtained by centrifugal washing in (i) was suspended in the l, and reacted at 6-10 ° C. for 18-20 hours with gentle stirring. After the reaction, the liposome suspension was washed by centrifugation to remove unsensitized protein solution, and resuspended in 1 ml of 0.1% NaN ₃ -containing gelatin veronal buffer (pH 7.4).

(3) Measurement of anti-ATLV antibody using ATLV-related antigen-sensitized liposome: Measurement was performed using a 96-well microplate (Sumitomo Bakelite). 0.5m for dilution of liposome and sample
A gelatin veronal buffer (GVB ²⁺ ) containing M MgCl ₂ and 0.15 mM CaCl ₂ was used. The ATLV-related antigen-sensitized liposome prepared in (2) was diluted 400-fold with GVB ²⁺ to 50 μl,
Immobilized human serum diluted appropriately with GVB ²⁺ 2
5μ was added. Subsequently, 25 μm of guinea pig complement was added and reacted at 37 ° C. for 2 hours. Reaction is 10 mM EDTA
The suspension was stopped by adding 100 μl of a peronal buffer solution (pH 7.5). The amount of CF transmigration depending on the ATL antibody titer was measured with a fluorescence-side fixed device (excitation: 490 nm, fluorescence: 530 nm).

FIG. 1 shows a standard curve obtained by assembling dilution series of ATL antibody-positive specimens and assaying as described above. As is clear from FIG. 1, CF release depending on the amount of antibody was observed, and when the amount of complement was changed from 1.5 to ₅ CH50 / ml and assayed according to the above, the sensitivity was dependent on the amount of complement. Rose.

(4) Reaction time course: The reaction time course was examined using ATLV-related antigen-sensitized liposomes. That is, ATL antibody-positive specimens were 40-128
It was diluted 0-fold and the complement concentration was 5CH ₅₀ / ml. The assay was performed according to (3), and the reaction was examined up to 180 minutes. The results are as shown in FIG. 2. The CF release increased depending on the reaction time, but it was found that there was no problem in the measurement sensitivity if the CF release was 2 hours or more.

(5) Comparison between the present measurement method and the conventional method: The comparison between the present measurement method and the conventional method (aggregation method) was performed for 15 samples. The value of the conventional method is represented by the highest dilution factor of the sample to be aggregated, and the standard method is represented by the highest dilution factor of the sample having a CF release of 5% or more. The aggregation method was started with a two-fold dilution, and the measurement method was started with a ten-fold dilution. The results are as shown in Table 1, and show that the conventional method is satisfactory as an ATL solid measurement method.

[Brief description of the drawings]

FIG. 1 shows a standard curve when the ATL antibody in the test serum was measured using the ATL antibody measurement reagent of the present invention, and FIG. 2 shows the measurement of the ATL antibody in the test serum using the reagent. It is a figure which shows the reaction time course at the time of doing.

Claims (1)

(57) [Claims] 1. An ATLV-related antigen solubilized with sodium deoxycholate is bound to the surface of a liposome containing phospholipids and cholesterol as main components via a crosslinking agent, and a hydrophilic labeling substance is present in the liposome. An ATL antibody measuring reagent characterized in that