JPS61176855A - Reagent for immune analysis and immune analysis method - Google Patents

Abstract

PURPOSE:To decrease considerably the measuring error arising from a sample by forming a reagent for immune analysis of a microspore having a film labeled by an antigen or antibody and contg. internally a chelate agent which colors when coordinated to metallic ions. CONSTITUTION:The microspore consisting of, for example, a spherical particle formed of a lipid thin film and the film is labeled with the isogenic antibody if the measuring object is an antigen and is labeled with the isogenic antigen if the measuring object is an antibody. The microspore contg. the chelate agent is obtd. by dispersing the lipid into a soln. prepd. by dissolving the selected chelate agent to a prescribed concn. to form the many particles contg. the chelate agent and washing away the unnecessary matter therefrom. An antigen- antibody reaction arises between the antibodies or antigen labeled to the film of the microspore and the co-existing complement is activated to attach and dissolve the microspore film when the microspore is made to exist with the sample for measuring the antigen or antibody. The chelate agent contained in the microspore is then liberated and reacts with the metallic ion existing on the outside of the microspore so as to form a color.

Description

Detailed Description of the Invention [Field of Application of the Invention] The present invention relates to immunoassay reagents and immunoassay methods.
In particular, the present invention relates to immunoassay reagents and immunoassay methods that utilize endoplasmic reticulum with membranes labeled with antigens or antibodies.

[Background of the invention]

A typical immunoassay method that has been put into practical use so far utilizes an antigen-antibody reaction between an antibody labeled with a radioisotope and an antigen in a sample collected from a living body to detect the antigen in the sample. This is a radioimmunoassay method for quantitative analysis. However, since this radioimmunoassay method is cumbersome to handle, a simple method has been proposed in which the endoplasmic reticulum is used for quantitative analysis using an absorptiometer. Japanese Patent Application Laid-Open No. 56-132564 describes a method for analyzing this food, using a lipid molecule membrane as an endoplasmic reticulum or liposome.
It has been proposed to quantitatively analyze antigens or antibodies using enzymatic reactions by carrying an enzyme within the endoplasmic reticulum and allowing a substrate that exhibits immune-specific attack to exist outside the endoplasmic reticulum.

JP-A-59-2 as another example of this type of analysis method
No. 8661 discloses an immunoassay method in which the cell membrane of a sheep red blood cell is used as the endoplasmic reticulum, an enzyme or a substrate is housed in the endoplasmic reticulum, and an enzyme reaction is similarly utilized. In either method, the membrane of the endoplasmic reticulum is labeled with an antigen or a cognate antibody.

However, these methods using enzymatic reactions have the problem that measured values vary depending on the sample and measurement errors are large.

[Purpose of the invention]

An object of the present invention is to provide an immunoassay method that can reduce measurement errors even if a sample contains a substrate or an enzyme, and an immunoassay reagent used therein.

[Summary of the Invention] The present invention provides that, when a substrate or enzyme is present in a serum sample, the enzyme or substrate released from the endoplasmic reticulum due to the antigen-antibody reaction may be combined with the substrate or enzyme derived from such biological sample. This was done with the focus on the fact that this reaction often results in large background and reduces measurement accuracy.

The first invention has a membrane labeled with an antigen or antibody,
An immunoassay reagent comprising a endoplasmic reticulum containing a chelating agent that develops a color when coordinated with a metal ion is provided.

In the second invention, an endoplasmic reticulum having a membrane labeled with an antigen or an antibody, a chelating agent that is retained within the endoplasmic reticulum and develops a color when coordinated with a metal ion, and the endoplasmic reticulum are dispersed. and a liquid prepared so that the metal ions are present outside the endoplasmic reticulum.

Furthermore, a third invention provides a sample containing an endoplasmic reticulum having a membrane labeled with an antigen or antibody and containing a chelating agent therein, a metal salt capable of reacting with the chelating agent, complement, and the antibody or antigen. and causing the chelating agent to flow out of the endoplasmic reticulum by the antigen-antibody reaction that occurs;
and quantifying the antigen or antibody in the sample by measuring the reaction product of the chelating agent and the metal salt.

Since the chelating agent is not derived from biological samples, the same level of background is obtained for all samples.
Since the background itself is small, the S/N ratio is improved.

[Embodiments of the invention]

The endoplasmic reticulum is composed of, for example, a spherical particle formed of a thin lipid film, and this membrane is labeled with a cognate antibody if the target to be measured is an antigen, or a cognate antigen if the target to be measured is an antibody.

Preparation of such endoplasmic reticulum is carried out by the method of ) (Sia et al. (Me
thod in ]i:nzymo1gy-, vot.

73.152 pages) t-Applicable. The endoplasmic reticulum containing the chelating agent disperses lipids in a solution in which the selected chelating agent is dissolved at a predetermined concentration to form a large number of particles containing the chelating agent, and then washes and removes unnecessary substances. obtained by. The particle size of the endoplasmic reticulum is preferably 1 mμ
~1 ml, but may exceed this range. As a chelating agent, 2-5-Br-2 pyridi#7zo5-Npropyl-N sulfopropylaminoaniline (hereinafter referred to as 5-Br-PAP 8) ), dimethylsulfonacy-m, chromazurol B.

TAM8MB, nitroso-PSAP, bathophenandroline (BFT), etc. can be used. When these chelating agents are coordinated with a metal ion, particularly a heavy metal ion, the chelate compound develops a color, which enables optical measurement using a photometer.

As the metal salt to be present in the solution mixed with the sample when causing the antigen-antibody reaction, salts of iron, aluminum, magnesium, calcium, nickel, cobalt, etc. can be used. When the endoplasmic reticulum is present with a sample for measuring an antigen or antibody, an antigen-antibody reaction occurs between the antibody or antigen labeled on the endoplasmic reticulum membrane, and the coexisting complement is activated and attacks the endoplasmic reticulum membrane. and dissolve. As a result, the chelating agent housed within the endoplasmic reticulum is released, reacts with metal ions outside the endoplasmic reticulum, and changes color depending on the extent of the chelating agent released. Since the amount of antigen or antibody in the sample is proportional to the number of endoplasmic reticulum destroyed, the concentration of antigen or antibody corresponding to the degree of coloration can be determined from a previously determined calibration curve.

Next, the experimental row will be explained.

A liquid used as the internal liquid phase of the endoplasmic reticulum is prepared so that the chelating agent 5-Br-PAPS has a concentration of 10 mate/L. A lipid that can be labeled with an antigen or an antibody is dispersed in this liquid to obtain a large number of particles containing a chelating agent. The outer surface of the granules is cleaned by repeatedly rinsing off the granules other than the obtained granules. After this cleaning, '[(
Thyroxine according to the method of Sia (supra)
The antigen (hereinafter referred to as T4) is bound to the endoplasmic reticulum membrane and labeled. The cells thus prepared can also be placed in a container together with a buffer and used as a reagent for immunoassay. Alternatively, the endoplasmic reticulum can be dried and provided in the form of granules or tablets for analysis reagents.

0.001- of the endoplasmic reticulum obtained as described above was
．． Ferrous chloride (FeC42) was dispersed in an isotonic solution consisting of 154 mot/t of sodium chloride, and 10% of ferrous chloride (FeC42)
After adding 0mM and adding 20μt't of complement, the total amount was 1t
And so. This reagent solution can be stored in a container and used as an immunoassay reagent if necessary.

Set the sample container containing the serum to be the specimen into the sampler of the clinical automatic analyzer. The reaction line is heated to 37C, and the reaction vessel rows are transferred intermittently. A sample dispensing mechanism, a reagent supply mechanism, and a multiwavelength photometer are installed near the reaction line.The sample dispensing mechanism collects the serum sample from the sample container and transfers the sample to the reaction container on the reaction line. Supply 10 μtk. 2 d of the reagent solution obtained as described above is added to the transferred reaction container by the reagent supply mechanism. Thereafter, the reaction vessels are incubated while being transferred, and after 30 minutes, the reaction vessels containing the reaction solution are brought to the photometry section. Irradiate the reaction solution with light at a wavelength of 552n.
The absorbance at m is measured, the measurement result is calculated by the control calculation section, and the concentration of anti-T4 antibody contained in the sample is printed out by a printer.

Figure 1 shows a calibration curve series. This calibration curve was prepared using the above-mentioned reagent solution, serum without antibodies, and anti-T at a predetermined concentration in a reaction container.
4 antibodies were added. The sample blank value has an average value of 0.
．． The dispersion was 0.02 μM/l. The reproducibility when the average antibody concentration was 15 μM/l was 0.02 μM/l with a standard deviation. The sensitivity of this reaction system at serum level was 2×10′″aM/l.

〔Effect of the invention〕

As explained above, according to the present invention, measurement errors originating from the sample can be significantly reduced, so the measurement accuracy of immunoassays can be improved, and ultra-trace analysis is also possible.

[Brief explanation of the drawing]

2shi, xo, yo dσPpaI
N T...

Claims (1)

[Scope of Claims] 1. An immunoassay reagent comprising a endoplasmic reticulum that has a membrane labeled with an antigen or antibody and contains a chelating agent that develops a color when coordinated with a metal ion. 2. An endoplasmic reticulum having a membrane labeled with an antigen or antibody, a chelating agent that is retained in the endoplasmic reticulum and develops a color when coordinated with a metal ion, and an endoplasmic reticulum in which the endoplasmic reticulum is dispersed. and a liquid prepared such that the metal ions are present on the outside of the reagent. 3. Mixing an endoplasmic reticulum with a membrane labeled with an antigen or antibody and containing a chelating agent inside, a metal salt capable of reacting with the chelating agent, complement, and a sample containing the antibody or antigen. , Flowing out the chelating agent from the endoplasmic reticulum due to the antigen-antibody reaction that occurs, and quantifying the antigen or antibody in the sample by measuring a reaction product of the chelating agent and the metal salt. Immunoanalytical methods including.