JPH03152467A - Hbs antibody measuring reagent - Google Patents

Abstract

PURPOSE:To achieve automation of measurement being made highly sensitive, accurate and inexpensive with a simple operation by bonding a refined HBs antigen to the surface of liposome mainly composed of phospholipide and cholesterol or others through a crosslinking agent. CONSTITUTION:A measurement is performed with a simple automatic operation by a measuring reagent of a HBs antibody utilizing a complement dependent convergent liposome film damage reaction. Here, a refined HBs antigen is bonded to the surface of liposome mainly composed of phospholipide and choles terol through a crosslinking agent while a reagent is used with a hydrophilic label substance sealed into the liposome. Then, the reagent and the complement are added to an object to be inspected to cause a bonding reaction between antigen-antibody and the complement. So, the label substance is released from the liposome in proportion to a reaction value and then, a determination is performed by an analysis method corresponding to the substance and, for exam ple, an amount of an antibody in the sample can be measured by a calibration curve prepared previously.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides a reagent for measuring II-s antibody, and more specifically, a method for measuring HO8 by simple automatic operation using a complement-dependent ribosome membrane damage reaction. 11 that can measure antibodies
8s antibody measurement reagent.

[Conventional technology]

The HBs antibody is the only antibody that has virus-neutralizing activity among the related antibodies that appear in the blood after hepatitis B virus infection, and also plays a role as a reinfection-protective antibody. Therefore, detection of tlBs antibodies not only serves as a marker for recovery and cure from hepatitis B, but also serves as an important indicator for understanding the immune status before and after vaccination.

Countercurrent immunoelectrophoresis (CI) is used to test for Has antibodies.
B), two-dimensional immunodiffusion (MO), passive hemagglutination (PIIA), immunoadhesive hemagglutination (IAII8)
, enzyme immunoassay (BIA), solid-phase radioimmunoassay (formerly ^), and the like are known.

[Problem to be solved by the invention]

Among these, CIB and MO have long measurement times and low sensitivity. Furthermore, for PIIA and IAII8, it is difficult to prepare stable antigen-coated red blood cells, and the reagent costs are high.

Furthermore, the 81^ and R1 formulas are 5,000 times more sensitive than MO.
0 to 50,000 times, but the reagent price is high and R1
Because the formula uses radioactive elements, special radioactive element processing facilities are required.

Furthermore, in view of the clinical importance of I[]s antibodies as described above, automation of the measurement method is desired.

Accordingly, an object of the present invention is to provide a reagent for measuring 11Bs antibody that can be used to measure 1Bs antibody with high sensitivity, accuracy, and low cost through simple operations, and that can also automate the measurement.

[Means to solve the problem]

Under such circumstances, the present inventor conducted intensive research to solve the above problem, and as a result, discovered that the above object can be achieved by utilizing the complement-dependent ribosome membrane damage reaction, and has devised the present invention. completed.

That is, the present invention provides HBs, which is characterized in that a purified HBs antigen is bound to the surface of a ribosome whose main components are phospholipids and cholesterol via a crosslinking agent, and a hydrophilic labeling substance is encapsulated within the ribosome. The present invention provides antibody measurement reagents.

In the present invention, the ribosome may be any conventionally used ribosome as long as it has phospholipids and cholesterol as its main components, but when the ratio of phospholipids to cholesterol is around 1=1, it is stable. Ribosomes are easily obtained. Moreover, it is preferable that the fatty acid residue in the phospholipid has 12 to 18 carbon atoms, and more preferably an even number.

The labeling substance encapsulated within the ribosome must be hydrophilic and quantifiable when eluted outside the ribosome. 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 carboxyl fluorescein that emit very strong fluorescence at a temperature below M); Luminescent compounds such as luminol and luciferin that emit light by oxidation reaction outside the ribosome; 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 broken down by the action of oxidative enzymes, resulting in oxygen consumption or hydrogen peroxide production; Relatively large ionic compounds such as tetrapentylammonium; nicotinamide adenine Dinucleotide (NAD
Coenzymes such as ); radical compounds such as methyl viologen are desirable.

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

First, phospholipids and cholesterol are placed in a flask, a solvent is added and the mixture is allowed to react, and then the solvent is distilled off and dried by suction.

Thereafter, an aqueous solution of a predetermined labeling substance is added into a flask with a thin film formed on the wall, the flask is tightly stoppered, and the flask is shaken to obtain labeling substance-encapsulated ribosomes.

On the other hand, a cross-linking group is introduced by reacting Has antigen purified from It B s antigen-positive serum with a cross-linking agent in a buffer solution,
Thereafter, if necessary, the crosslinking group is further reacted with a reducing agent (e.g. dithiothreitol; TT),
Obtain modified antigen.

Finally, by reacting the labeled substance-encapsulated ribosome with the modified antigen in a buffer, an HDs antigen-sensitized ribosome, which is the reagent for measuring an HBs antibody of the present invention, is obtained. Such a II[ls antibody 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) phthalate (SMP), N-succinimidyl-4- (p-maleimidophenyl) acetate (SMP^), N-succinimidyl 4-(p~maleimidophenyl) propionate (SMPP), N-(
γ-maleimidobutyryloxy)succinimide (GM
[]S), N-(ε-maleimidocaproyloxy)succinimide (BMCS), and the like.

To measure Has antibodies in a specimen using the llBs antibody measurement reagent prepared in this way, the reagent and complement are added to the specimen to cause a binding reaction between antigen-antibody and complement. let Then, a labeling substance is released from within the ribosome 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 in the sample is measured using a calibration curve prepared in advance. Can be done.

The complement used in this measurement procedure 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 shown above, by using the Ill5 antibody measurement reagent of the present invention, it is possible to measure the flS antibody in a specimen with high sensitivity, accuracy, and low cost with simple operations, and it is also possible to automate the measurement. be.

〔Example〕

The present invention will be further explained with reference to Examples below.

Example I Preparation of Has antigen-sensitized ribosomes (1) Preparation of ribosomes Dipalmitoylphospha, tidylcholine (DPPC) 0
．． 5JJmoj2. 1. 2-Simyristoylamide-1,2-deoxyphosphatidylcholine (DDPC)
Q, 5μmoj! , Cholesterol (Chol) 1
μmo! ! and dithiopyridylated dipalmitoylphosphatidylethanolamine (DTP-DPPB) 0.
05 tz no 1 was placed in a pear-shaped flask, and the chloroform in which the lipids had been dissolved was distilled off using an evaporator. After further drying in a vacuum desiccator for 1 hour, 0.2M carboxyfluorescein (CF) was added to the pear-shaped flask.
0 μl was added, shaken vigorously, and the lipid thin film from the glass wall of the pear-shaped flask was peeled off to prepare CF-encapsulated ribosomes. Unsealed CF is 0. Centrifugal washing was performed three times with OIM II port [IS buffer (containing 0.15 M NaCj!, pH 7.5) for separation.

(2) Sensitization of Group 1 S antigen to ribosomes Subtype ad llBs antigen solution purified from serum (l'Bs, 0
．． 4mg/mf) 1.3mI! After performing ultrasonic treatment for 30 seconds (100 μA) using a probe-type sonicator, 13 μl of 30 mM SP leaf ethanol solution was added, and the mixture was allowed to react at room temperature for 30 minutes. Continued excessive S
To remove P, add 20mM MB5 buffer (0,
15M NaCj! Gel filtration was performed using Sephadex G-25 equilibrated with pH 6.0). Protein fraction obtained by gel filtration (2 ml of Centricon 10
After concentrating 4 times using DTTfc (manufactured by Amicon),
It was added to a final concentration of 100 mM and reacted at room temperature for 10 minutes.

0.0 to subsequently remove excess DTT. OIMII
B P [Sephadex G-2 equilibrated with IS buffer (containing 0.15 M NaCj!, pH 7.5)
5 to obtain a protein fraction of 1 rnl. Measure this protein fraction 1d using a probe sonicator for 30 seconds (
After ultrasonication (100 μA), the ribosome pellet prepared in (1) was added and reacted at 6 to 10° C. for 18 to 24 hours with slow stirring.

Thereafter, the ribosome suspension was centrifugally washed, unreacted proteins were separated, and resuspended in gelatin-heronal buffer (GVB-, pH 7,5) 1- containing 0.1% NaN5.

Example 2 Preparation of calibration curve 96-sized microplate (manufactured by Sumitomo Bakelite)
Measurements were made using 0 for ribosome and sample dilution
．． 5 mM MgCl, and 0.15 mM CaCl,
llBs antigen-sensitized ribosome prepared in Example 1 was used.
VB”te 500 times diluted 25uj!l:, IgG
20.00010/j!
(WIIO standard) was diluted several times and 25 μl was added, and the mixture was reacted at 40°C overnight or at 37°C for 2 hours. After that, 25 μm of appropriately diluted guinea pig complement and GVB”
25 ttl was added, and the reaction was further carried out at 37°C for 1 hour. The reaction was carried out using GVB- (pH 7) containing 10 mM BUT^.
, 5) to 100 μji! Additionally, it was stopped. The amount of CF depending on the amount of flBs antibody was measured using a microplate fluorometer MTP-32 (Corona Inc.) with an excitation light of 490 nm.
, fluorescence was measured at 530 nm.

A standard curve prepared from the above measured values is shown in FIG. In addition, in FIG. 1, LILA value (%) was calculated according to the following formula.

F7: CF amount F obtained when adding anti-11Bs antibody
GVB instead of adding two antibodies and guinea pig complement
Amount of CF obtained when adding 10% FX: CF obtained when adding lO% Triton X-100 instead of guinea pig complement Example 3 Comparison with conventional method The correlation with a measurement method using an antibody measurement reagent (LILA method) was investigated.

A total of 72 HBs antibody-negative specimens and HBs antibody-positive specimens were used.

(1) Measurement of large samples using the LILA method Measurements were performed using 96 large microplates, and GVB2+
was used. Add 25 μl of 500-fold diluted ribosome solution to
25 μl of non-mobilized human serum diluted 2 times with GV02'' was added and allowed to react overnight (16 to 20 hours) at 4°C. Then, 10 Cl of GVB''25p 1 and GVB'+ were added.
Add 25 μl of guinea pig complement diluted 50 times, and add 3
The reaction was carried out at 7°C for 1 hour. The reaction was carried out using 10% GVB-(p)17.5) containing 5% sodium benzoate and 10% BDTA.
Added 0 μl and stopped. Measurement of fluorescence intensity was performed according to Example 2. In addition, the LILA value (%) is based on the amount of CF liberated with 10% Triton expressed as relative fluorescence intensity.

(2) Measurement of a large sample using the PII 8 method For the large sample used for measurement in (1), perform a quantitative test of HBs antibody titer using Rafacel anti-HBs (manufactured by Hinaga Pharmaceutical Co., Ltd., P11 8 method) according to the package insert. I did it.

(3) Correlation between LILA method and PI (A method) Based on the measured values obtained in (1) and (2) above, LILA method and PHA method
Correlation Ma) An IJ hook was prepared and shown in Figure 2.

[Brief explanation of the drawing]

Figure 1 is a standard curve when Has antibody in a sample is measured using the Has antibody measurement reagent of the present invention, and Figure 2 shows the correlation between the measurement method using this reagent and the PHA method. It is a drawing. HBs antibody 1 degree (IU/1) Cause 1 Figure 2

Claims (1)

[Claims] 1. A reagent for measuring an HBs antibody, which is characterized in that a purified HBs antigen is bound to the surface of a ribosome whose main components are phospholipids and cholesterol via a crosslinking agent, and a hydrophilic labeling substance is encapsulated within the ribosome.