JPS6079268A - How to measure antigen-antibody reaction - Google Patents

Abstract

PURPOSE:To enable the treatment of large amount of samples for a short time by a method wherein a sample with unknown concentration of an antigen or antibody is made to react with an antibody or antigen and changes with time in the producing speed of the bond reactant obtained are measured. CONSTITUTION:A non-soluble carrier particle with a certain average size is sensitized with a certain antibody or antigen to prepare a sensitized carrier. The same antigen or antibody the same as those contained in a sample liquid to be measured actually to prepare standard sample liquids with various concentrations containing it in a liquid medium virtually the same as the medium of the sample liquid at various known concentrations. Then, the sensitized carrier is mixed with a standard sample liquid and the permeability of the reactant mixture is measured with the passage of time when the proceeding of the antigen- antibody reaction becomes stable. A corresponding curve is made up between the concentration of antigen or antibody in the standard sample liquid and the decrease rate (reaction speed) per unit time of the permeability of the reactant mixture. Then, the reaction speed is measured concerning a sample liquid with unknown concentration in the same way and compared to the corresponding curve thus obtained.

Description

[Detailed description of the invention] The present invention relates to a method for measuring antigen-antibody reactions.

Anti-fjaWr immunoassay, which utilizes body reactions, has recently seen rapid progress. This immunity ii
+: 1 regular method, divided into marked method and non-t1 method.Hino 1.
Ru.

Labels used in labeling methods include isotopes, fluorescent substances, electron spin resonators, luminescent materials, Kinho, bacteriophages, electrochemically active containers, etc., and labeled antigens or To determine the Uaj of 19 bodies, electron spin resonance, polarography, etc. are used for optical analysis of radioactivity, absorption, fluorescence, emission, etc.

On the other hand, in the non-labeling method, for example, antigens can be
Methods that optically capture changes in turbidity associated with agglutination reactions using tightly bonded insoluble particles (such as 2DEX), immunoturbidimetry using insoluble particles, and laser nephelometry are known. ing.

All of these immunoassay methods fail in that they either immerse or indirectly detect the light of a compound produced by the reaction between an antigen and an antibody.

However, in such an immunoassay method, there is a problem in measurement in the so-called "antigen excess region" where the antigen is present at a concentration higher than a certain level.

In particular, an example of the above-mentioned non-static method, Yang@(de), will be explained.

For example, when an antibody or antibody supported on insoluble conjugate particles is reacted with an antigen or an antibody in a liquid medium, the transmittance of the reaction mixture decreases as the reaction progresses (i.e., the absorbance decreases). The rate of the antigen-antibody reaction is measured from the increase), and the concentration of the antigen or antibody in the subject is determined from that rate.
There are known methods for determining According to this method, the concentration of antigen or antibody can be determined rapidly and with high accuracy.

However, for example, in the case of an insoluble carrier sensitized with an antibody, in a region where the anti-adhesive molecules are excessive compared to the antibody molecules, the blade wheel naturally contributes to particle aggregation. When this happens, the antigen-antibody reaction apparently progresses by 1 llq. A phenomenon known as the so-called anti-cool sea bream excess region is observed, and in such cases There often exists a number of concentrations corresponding to one reaction rate.

In clinical tests, the frequency of appearance of the above-mentioned non-labeling method r (+meter, antibody q that reduces the above-mentioned antigen excess area) is 1l-i
It was common practice to dilute the specimen beforehand and use it for testing.

However, with the advent of automatic machines, the technology for detecting these antigen-excess samples, which are clinically rare even though they are extremely small, has increased when processing large samples in a short period of time. It's necessary.

For example, in order to perform accurate measurements in such a case, it is necessary to always use a single dilution method or the like to carry out measurements twice or more on the same specimen, using a dilution method.

Therefore, the present inventors conducted various studies in order to find a measurement method more suitable for rapid processing of a large number of samples through automation.
We have arrived at the present invention.

That is, the gist of the present invention d, Antigen and antibody are reacted, and the reaction mixture is called tang.

d of antigen or antibody by measuring the biomass or rate.
In the immunoassay method for determining the centrality, a corresponding curve between the side value of the positive or rate of production of a reactive conjugate and the gHH degree, which is conducted using a sample with a known concentration of antigen or antibody, When the roughness of the sleep corresponds to one measurement 1il'I, the concentration of antigen or antibody in an unknown sample is determined from this corresponding curve. (1) A sample with unknown concentration of antigen or antibody, Determine the time change in the production rate of the reaction conjugate obtained by reacting with the antibody or 1.1c Ryo corresponding to the filji, and determine whether or not there is a Keyakita value (VrpaX) of the production rate. A 1'1'' standard is set for the concentration measurable region that can uniquely determine the concentration2, and (If) when the above maximum value (Vmax) is present, it is determined that it belongs to the νh degree measurable region7.
, 76 of the antigen or dW1 body in the unknown sample from the manual correspondence curve.
・It is a method of measuring the antigen-antibody reaction that determines the degree of

The present invention will be explained in detail below.

First, the immunoassay method t to which the present invention is applied is not particularly limited, and can be applied to both the above-mentioned labeling method and the imitation nine method.

For example, a typical method is radioimmunoassay (
RIA), enzyme immunoassay (KIA), photoimmunoassay, latex agglutination reaction, and immunoturbidimetry.
do.

In one embodiment of the present invention, an antibody or an antigen is supported on cold-bathed carrier particles, and the supported antibody or antigen is reacted with the antigen or antibody in a liquid medium to react with the reaction mixture. A case will be described in which the present invention is applied to a method (including a latex aggregation reaction) in which light is irradiated at a point of time equal to or higher than the surface hvt- and the decrease in transmittance of the reaction mixture is measured within a certain period of time.

First, in this method, insoluble carrier particles with an average particle size of about 6 μm or less, preferably 7.0 μm or less, are used, and the antibody or antigen is made to coexist (sensitized) with the analyte. The antigen or antibody in the reaction mixture is reacted, and the transmittance of the reaction mixture is set to 1, usually 0.3 to 2. ri μ, preferably θ, 6~/
The concentration of the antigen or antibody in the subject is determined by measuring the reaction rate with light having a wavelength in the μ range of 911.

Insoluble phase particles include organic molecules that are substantially insoluble in the liquid medium used when carrying out the measurements and have the above-mentioned average particle size, such as those produced by emulsion polymerization, such as polystyrene, styrene-butadiene copolymers, etc. the resulting latex,
Alternatively, an inorganic oxide such as alumina may be used.

Insoluble carrier particles (preferably Latinx particles) such as these are made to support (sensitize) antibodies or antigens that can react with the antigens or antibodies in the specimen to be measured by a conventional method. .

The concentration of insoluble carrier particles sensitized with antibodies or antigens is usually θ
, θ/heavy protein% or more, good! t2〈θ, /−/about 1.nl by weight of the suspension and (2×1.nl:)f.

This sensitized carrier is reacted with an antigen or antibody in a liquid medium under certain conditions, and the amount of decrease in permeability of the reaction mixture per unit time is measured after a certain period of time after the start of the reaction. can be quantitatively measured. It is desirable to measure this rate of decrease after the reaction between the sensitized carrier, which is a component of the reaction mixture, and the antigen or antibody in the test solution, and after the progress of the antigen-antibody reaction is at least stable. b
o For this purpose, mix the sensitized carrier and the test solution to a suitable size, or mix them under stirring, and measure their permeability, for example 2 to 3 seconds after mixing. It is suitable to measure.

Measurement of such antigen-antibody reactions is carried out, for example, as follows.

First, insoluble carrier particles VC having a certain average particle size
A sensitized carrier is prepared by sensitizing a certain antibody or antigen. On the other hand, using the same antigen or antibody as that contained in the test liquid (sample) to be actually measured,
Various concentrations of standard test solutions are prepared containing it at known concentrations in a liquid medium substantially the same as that of the test solution.

Next, the sensitized carrier and the standard sample solution are mixed together, and when the progress of the antigen-antibody reaction is stabilized, the transmittance of the reaction mixture is measured over time. For example, at the stage where the transmittance decreases quantitatively, Mil
The rate of decrease in transmittance of each reaction mixture per unit time is measured for the test liquids at various concentrations.

Next, the rate of decrease in , for example, is plotted on a graph with the concentration of the antigen or antibody in the standard test solution +t as the axis and the rate of decrease as the vertical axis.

A corresponding curve between the antigen or antibody concentration in the standard test solution and the rate of decrease (reaction rate) in permeability of the reaction mixture per unit time is obtained.

Therefore, for a specific antigen or antibody, create a corresponding curve as described above. Then, by measuring the same reaction rate as above for flk 11 (unknown) test solution containing the same antigen or antibody, and comparing this with the corresponding curve, the test solution can be determined. The amount of antigen or antibody contained in the protein is regularly measured [2].

The present invention uses the above measurement method at or-t, -+ and at a concentration of 1M.
, based on the correspondence curve between the reaction rate and Shibukawa's obtained by m-sample of knowledge, we found that one reaction rate corresponds to Shosho's? 1' is useful for determining the concentration of an unknown sample from this correspondence curve.

For example, in order to measure the reaction rate and determine the efficiency of a reaction with an unknown concentration of antigen (1), first, By the 4111 constant, the corresponding curve of anti-Q ceramic M [and the reaction rate is found] 1
That's what I'm doing.

(2) It is necessary to have the reaction rate of the unknown sample y and its corresponding curve force, h, which can uniquely determine the concentration. As the antigen concentration increases, the reaction rate also increases, but a region appears where the rate of increase in the reaction rate decreases, and as the antigen concentration increases further,
A region where the filtration reaction rate decreases may appear, or after decreasing, it may increase or decrease again.

In such a case, since a plurality of concentrations correspond to one reaction rate, the concentration of the unknown sample cannot be uniquely determined. In this way, in the area where antigen overload occurs, it is difficult to expect aggregation reactions and changes in permeability due to the presence of the antigen target. First of all, it is necessary to determine whether or not it exists.

I can do it. If it is determined that the concentration is in an area where it is impossible to measure the concentration, the sample can be diluted and measured in a measurable area.

Therefore, we will further explain the measurement method of the present invention in the latex agglutination reaction described above.

First, take a sample whose antigen or antibody base IW is unknown.

The time change in the rate of production of a reactive conjugate obtained by sensitizing the corresponding antibody or antigen and reacting with a latex reagent is determined as n+++. Nomatao with fr and λ.

(for example, 30 seconds after the reaction starts), 81
K) Tae) et al., Transmission at a certain time 1 j7. ．． Add the rate attj at an interval selected from about /~gθ seconds, -
The next day +: (J: J, t- = the thickness of the slope at the initial rate, lc+-J, the product reaction rate is favorable. The average reaction rate near +, -, etc. of one point were measured: ii, 7+.

The present invention is based on the presence or absence of this extremely high production rate (Vmax), which can uniquely determine the anchorage of antigens or antibodies in unknown samples.
, whether or not to do i'IQ to I.1If1 Castle is clearly stated in Threat Heading 7, Book).

The range of degrees where this extremely thick value (Vmax) exists, l
Although it depends on the latex particle size, temperature, measurement wavelength, speed acquisition interval, etc., these conditions should be selected appropriately to obtain the clinically necessary concentration range. I can do it.

The above VmaX exists; 2. When it can be determined that the concentration is within the normal range, the concentration of the antigen or antibody in the unknown sample can be determined from the above-mentioned corresponding angle.

In this case, a corresponding curve was created for samples with known antigen or antibody concentrations (111. Daibutsu Vmax and ρ
Using the correspondence curve with the concentration, it is possible to determine the concentration for even higher concentration ranges.

On the other hand, when the extremely thick value Vmax does not exist, it is determined that r, 5 is not within the measurable silver degree range, and trial 1 can be diluted and further provided as #W n+1+ constant.

The present invention is not limited to the above-mentioned standard immunotherapy method, but can also be applied to other standard immunotherapy methods in which the anti-restriction surplus is a problem.

In the case of stiffness, 14-year-old speed is 117, which is commonly used in each of the 1st method! ! It can be obtained orally or indirectly from I-contact, and I! +＜Thick IllζVnl
The presence or absence of aX can be known.

The method for measuring an antigen-antibody reaction according to the present invention is particularly useful for JI+4 cases in which a large amount of specimens are collected in a short period of time as described above.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example/CAFP (α-fetoprotein) i11] Constant J measurement conditions (latex agglutination reaction) (a) Latex (LTX): Particle size θ1.2Yl
! m Accuracy 7% AFP sensitization←) Measurement system Standard material (Std, )! Dilute θμ Stabilizing solution and buffer Oθ/it Sensitized latex 2θ111 Wavelength θ, 9 μm (c) After the reaction starts as the initial point of speed measurement? 4fh f >> 4 h, za seconds 11'' I set the permeability a+:+ at J intervals, and investigated the value of 1.1, the value Vma, and the value of X.

The results are shown in Figures/~2. Vm and aX were obtained up to a concentration of approximately 0θθg/□, e (see 1).

It is determined that the concentration can be measured up to a concentration of θθθ+v/ml.

The particle size of the example coratex was 0. -2ltm, concentration 0.23
The presence or absence of the maximum value Vmax was examined for 0RP (C-reactive protein) K')IA in the same manner as in Example/, except that , θθθng/rnl was obtained.

[Brief explanation of the drawing]

Figures/7 are diagrams showing the relationship between the production rate (V) of permeated N5(T)1-reactive conjugate and time for AFPVc. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney: Hase 7) 17 people Figure 1 time (mjn,) Figure 3 IZ, J f/iyye (mity,) time and min) ・J spoon Akira °1 - Nyama J, LIE 'tE;E' (
) j j () 11 ((Japanese 5) 9 i December % # - 11 1 small i' 1 indication / I > j 2 Title of invention 1 lill method of antigen-antibody reaction d, 33 Case of person who submits amendment What relationship is the special applicant (!+96) Mitani Kasei [- Industry Co., Ltd. 14 agent 〒1
0f) 2-1 Marunouchi, Chiyo 1.11-ku, Tokyo:] No. 2 Mitsubishi Kasei 21 Industry Co., Ltd. (1 person) 5 Amendment Order Roy q 1982 f411-j 31
El (light i;!1lJ) 6 For application subject to correction, light l1ll i,! ；Upper drawing 7 Contents of amendment

Claims (1)

[Claims] (1) In an immunoassay method in which the concentration of an antigen or antibody is determined by reacting an antigen with an antibody and measuring the production polarity or rate of the reaction conjugate, the IIA degree of the antigen or antibody is known. In the correspondence curve between the measured value and the concentration of reaction conjugate production rate obtained using a certain sample, if multiple concentrations correspond to one measuring candy, it is possible to determine the antigen in the unknown sample from this correspondence curve. Or pregnancy testing to determine the concentration of antibodies: (1) Measuring the time change in the production rate of the reactive conjugate obtained by reacting a sample with an unknown concentration of antigen or antibody with the corresponding antibody or antigen. , the extreme value of production rate (V+n
(g) When the maximum value (Vrnax) exists, g degree 81IJ
It is determined that it belongs to the configurable area. Determining the concentration of antigen or antibody in an unknown sample from the upward rudder correspondence curve, the antigen-antibody reaction consists of various! If! l determination method.