JP2510551B2 - Method for measuring antigen-antibody reaction - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring an antigen-antibody reaction.

(Problems to be Solved by Conventional Techniques and Inventions) Conventionally, an antibody or an antigen supported on insoluble carrier particles such as latex is reacted with an antigen or an antibody in a liquid medium, and a reaction mixture accompanying the reaction progresses. Decrease in transmittance,
That is, a method is known in which the rate of the antigen-antibody reaction is measured from the increase in absorbance, and the concentration of the antigen or antibody in the sample is quantified from the rate (described in Japanese Patent Publication No. 58-11575). By this method, the concentration of the antigen or antibody can be rapidly quantified with high accuracy.

However, in this method, it is possible to obtain a reliable measurement value only at a concentration of about several ng / ml in terms of sensitivity,
Items such as growth hormone, insulin, and digoxin that require more sensitive measurement were not always satisfactory. Further, the conventional measuring method using insoluble carrier particles such as latex has a drawback that it is easily affected by a coexisting substance existing in the reaction system when measuring an antigen or an antibody in serum or the like.

(Means for Solving Problems) Therefore, the present inventors have conducted diligent research to find a measurement method that enables measurement of a lower concentration of antigen or antibody and is not affected by coexisting substances in the reaction system. The present invention has been reached.

That is, the gist of the present invention is to react unreacted magnetic latex and antigen-antibody reaction carried on the magnetic latex after reacting the antibody or antigen carried on the magnetic latex with the antigen or antibody present in the liquid solvent. The substances are collected by applying a magnetic field to remove the liquid solvent, and then an eluent is added to elute the antibody carried on the magnetic latex or the antigen or the antibody or the mixture thereof which has reacted with the antigen, and then again. The magnetic latex is collected by applying a magnetic field to separate the eluate containing the eluted antigen or antibody or a mixture thereof from the magnetic latex, and the eluate is adjusted to a pH condition suitable for the antigen-antibody reaction, The insoluble carrier particles carrying the antibody or antigen are dispensed and reacted, and the degree of aggregation of the reaction mixture is optically determined by the intensity of transmitted light or scattered light. Reduction, or consists in the measurement method of the antigen-antibody reaction, wherein the capture visually.

 Hereinafter, the present invention will be described in detail.

In the method of the present invention, an antibody or antigen supported on a magnetic latex is reacted with a corresponding antigen or antibody in a liquid medium, and then the magnetic latex and the liquid solvent are separated by applying a magnetic field to remove the liquid solvent.

Then, an eluate is added to elute the antigen or antibody or the mixture thereof which has reacted with the magnetic latex. Then, a magnetic field is applied to the eluate to collect the magnetic latex, and the eluate and the magnetic latex are separated and removed. Next, this eluate is adjusted to a pH suitable for the antigen-antibody reaction, and then reacted with insoluble carrier particles such as latex carrying (sensitized) antibody or antigen to optically determine the degree of aggregation of the reaction mixture. Alternatively, measure visually.

The magnetic latex used in the present invention is granular,
Plates and the like are usually adopted.

Examples of the antigen to be measured and reacted in the present invention include various substances such as proteins, polypeptides, steroids, polysaccharides, lipids and pollens. Examples of the antibody include proteins that are antibodies against the above-mentioned antigens. A method for supporting an antibody or an antigen on the magnetic latex can be a conventional method.

That is, an antibody or an antigen capable of reacting with an antigen or an antibody in a subject may be physically adsorbed, or chemically bound using a coupling agent or the like. The reaction between the magnetic latex carrying (sensitized) the antibody or the antigen and the antigen or the antibody in the liquid medium is usually about 5 to 30 minutes after thoroughly stirring, although it varies depending on the kind. The reaction temperature is usually room temperature, but in order to shorten the time, the reaction may be performed at about 30 ° C to 40 ° C.

Separation of the magnetic latex that has reacted with the antigen or antibody in the liquid medium from the liquid medium, and separation / removal of the magnetic latex from the eluate after elution of the antigen and antibody are performed by applying a magnetic field using a magnet. It can be carried out by a method of settling and separating the latex.

The most commonly used eluent for eluting the antigen or antibody (antigen-antibody reaction product) that has reacted with the antibody or antigen carried on the magnetic latex is 0.2M glycine / HC1 buffer (pH 2.3 to 3.0). used. In addition, phosphoric acid-citric acid (pH 2.8), 1M propionic acid, 3M NaSCN (pH 7.5) and the like are used. The amount of the eluate is not particularly limited, but it is preferably smaller than the amount of the liquid medium to be measured in order to have the concentration effect. This volume is determined by the desired concentration ratio. The reaction time for elution is usually about 5 minutes
Equilibrium is reached in 20 minutes.

After elution of the antigen or antibody, the magnetic latex is removed from the eluate by the above-mentioned separation means, and then the eluate containing the antigen or antibody is adjusted to a pH of usually 6 to 9 with, for example, a trisaminomethane buffer.

The insoluble carrier particles used in the present invention have a particle size of 0.
Latex particles from 01μ to 1μ are optimal, usually 0.001
It is used as a suspension of insoluble carrier particles in an amount of not less than wt%, preferably about 0.01 to 1 wt%. The insoluble carrier particles are sensitized with an antibody or an antigen, and this is allowed to undergo an agglutination reaction with the antigen or the antibody in the eluate adjusted to about pH 6 to 9 described above. The degree of agglutination can be optically quantitatively captured as a change in intensity of transmitted light or scattered light. Alternatively, the degree of aggregation can be visually recognized by reacting the antigen or antibody in the eluate with the antibody or antigen carried by the insoluble carrier particles on the slide.

By the method described above, by concentrating the target antigen or antibody from the measurement liquid medium and separating the measurement liquid medium and the target antigen or antibody, without being affected by coexisting substances contained in the measurement liquid medium. The concentration of the antigen or the antibody in the measurement liquid medium can be accurately measured by using the calibration curve of the antigen or the antibody prepared from the sample of which the concentration is known in advance.

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist.

Example 1 Measurement of trace amount of AFP (alpha-fetoprotein) in serum As a solid phase, a magnetic latex having an average particle size of 0.7 μm (manufactured by Rhone Poulenc) was used.

First, sensitize AFP to 1% V / V magnetic latex. Dispense 260 μl of AFP positive sample and 40 μl of 1% V / VAFP magnetic latex into a test tube, and stir for about 1 minute. Approximately 10 minutes after stirring, place a magnet on the wall of the test tube and collect the latex that has undergone agglutination reaction with AFP. After confirming that the solvent and the latex are completely separated, the supernatant is removed. Next, add 300 μl of water to the test tube, stir, collect the latex again with a magnet,
Remove the supernatant and wash. The aggregated latex remaining on the wall of the test tube was washed with 100 μl of pH 2.3 glycine-HCl buffer.
And stir well to disperse to elute the AFP antigen.
The magnetic latex that has been eluted is collected by a magnet and the supernatant 10
Dispense 0 μl into the reaction cell, buffer 50 μl, stabilizing solution 110
μl [Mitsubishi Chemical Industries (currently Mitsubishi Chemical Corporation),
Immunoassay device “LPIA 100” for AFP measurement] is mixed, and thereafter, immunoassay device “LPIA 100” [manufactured by Mitsubishi Kasei (now Mitsubishi Kagaku)] AFP latex 40 μl is dispensed and stirred,
The reaction rate is measured. FIG. 1 shows a calibration curve of reaction rate V and AFP concentration.

(Effect of the Invention) According to the method of the present invention, it is possible to concentrate a trace amount of an antigen or antibody in a sample, which could not be measured by an immunoassay method using insoluble carrier particles such as latex, to a measurable concentration. As a result, this can be measured, and highly accurate analysis that is not affected by coexisting substances in the sample can be performed.

[Brief description of drawings]

FIG. 1 shows the calibration curve (reaction rate V and AFP) obtained in Example 1.
It is a figure which shows the relationship of density.

Claims (2)

(57) [Claims] 1. An unreacted magnetic latex and an antigen-antibody reaction product carried on the magnetic latex after reacting an antibody or an antigen supported on a magnetic latex with an antigen or an antibody present in a liquid solvent. The liquid solvent is collected by applying a magnetic field to remove the liquid solvent, and then an eluate is added to elute the antibody carried on the magnetic latex or the antigen or the antibody or the mixture thereof reacting with the antigen,
The magnetic latex is collected by applying a magnetic field again, the eluate containing the eluted antigen or antibody or a mixture thereof is separated from the magnetic latex, and the eluate is adjusted to a pH condition suitable for the antigen-antibody reaction. After that, insoluble carrier particles carrying an antibody or an antigen are dispensed and reacted, and the degree of aggregation of the reaction mixture is optically detected by intensity change of transmitted light or scattered light, or by visual observation. How to measure reaction. 2. The eluate from which the magnetic latex has been separated is adjusted to a pH condition suitable for an antigen-antibody reaction on a slide, and then insoluble carrier particles carrying an antibody or an antigen are added and reacted, and the degree of aggregation of the reaction mixture. The method according to claim 1, characterized by visually recognizing.