JPH03191863A - Enzyme immunoassay method - Google Patents

Abstract

PURPOSE:To make it possible to measure a physiological active material simply in a short time by making the physiological material comprising two subunits react at the same time with insoluble antibody and an enzyme-labeled antibody. CONSTITUTION:An insoluble antibody, an enzyme-labeled antibody and a sample are made to react under the coexistence of a monoclonal antibody which recognizes a bridge part wherein two subunits of other physiological materials are bonded. The enzyme activity of the enzyme labeled antibody which is bonded to the insoluble antibody or the enzyme activity of the enzyme-labeled antibody which is not bonded to the insoluble antibody is measured. Thus, the concentration of the intended physiological material comprising two subunits is measured. When one-step sandwich method is performed by using the antibody for the respective subunits of the physiological material comprising two subunits, the monoclonal antibody which recognizes the bonding parts of the two subunits of the other physiological material whose one subunit is common is made to exist. Thus the higher degree of the effect is avoided.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a method for measuring trace components in body fluids using antigen-antibody reactions, and in particular, the present invention relates to a method for measuring trace components in body fluids using antigen-antibody reactions, and in particular, when the target substance to be measured is a physiologically active substance consisting of two subunits. With a single immune reaction,
This is a method related to the so-called one-step enzyme immunoassay method for measuring, and can be widely applied to in vitro diagnostic drugs that measure various trace components composed of subunits.

(Relationship with Prior Art) Conventionally, there have been various methods for measuring trace components in body fluids using antigen-antibody reactions for physiologically active substances consisting of two subunits, which are trace components present in body fluids. do. In a method using a solid phase, an antibody against a subunit specific to the physiologically active substance is immobilized on the solid phase, and the physiologically active substance to be measured is labeled with an enzyme or a radioisotope, and then its constant There is a so-called competitive method in which a sample is mixed with a sample containing the target substance to be measured, washed and separated, and then the labeling activity of the labeled physiologically active substance bound to the solid phase is determined. However, this method has poor sensitivity due to poor precision at low concentrations, the measurement range is narrow, and the degree of purification of the target substance to be labeled affects the measurement system, so the purity of the target substance must be strictly controlled. There are drawbacks.

Still another method is to react the physiologically active substance with an insolubilized antibody that recognizes one subunit of the physiologically active substance bound to a solid phase, and then wash the system to remove other unreacted physiologically active substances. Next, a labeled antibody that recognizes the other subunit is labeled with an enzyme, etc., and a second reaction is carried out. After washing again, the activity of the labeled substance such as enzyme activity on the solid phase is determined, and the standard There is a method known as the so-called two-step Sanderch method, which measures the concentration from a curve. This method reduces the immune response by two
This process has to be carried out twice, and cleaning is also carried out twice, making the operation very complicated. Furthermore, it has the disadvantage that measurement takes a long time.

Therefore, in recent years, monoclonal antibodies using cell fusion technology have been used to overcome these drawbacks. When measuring with a single immune reaction using a monoclonal antibody,
If there is another physiologically active substance that shares a subunit in the measurement sample, the measurement value may not be measured correctly due to its influence.

Physiologically active substances consisting of two subunits, especially thyroid-stimulating hormone (TSH), luteinizing hormone (LH),
Follicle stimulating hormone (FSH), chorionic gonadotropin (
The hormone HCG) is composed of an α subunit and a β subunit, the α subunit being common to these hormones, and the β subunit being specific to the hormone.

When measuring these hormones using antibodies against the α subunit or monoclonal antibodies against the β subunit, the two-step sandwich method is used to avoid the influence of other hormones that share the α subunit. Generally measured. That is, the influence of hormones that share the α subunit, which are present in large excess, is washed away from the measurement system by washing, and then a labeled antibody is added to avoid the influence of the hormones that share the α subunit. When these hormones are measured using the one-step sandwich method, for example, when measuring TSH,
When HCG is present in large excess in the substance to be measured, such as in serum from pregnant women, the monoclonal antibody against the α subunit binds to HCG, reducing the amount of binding to TSH as the measurement target, and as a result, the TSH measurement value began to show a low value, and it was difficult to obtain an accurate value using the one-step Sanderutsch method.

(Problems to be Solved by the Invention) Even if an antibody that recognizes a specific subunit of a physiologically active substance consisting of two subunits and a monoclonal antibody that recognizes the binding portion of the two subunits of the hormone are used, It is believed that a one-step sandwich method can be used. However, in this case, due to the steric relationship between the recognition sites of each antibody, a certain amount of time is required for each antibody to bind to the hormone, so there is a problem that measurement time is required. The disadvantage is that for good measurements, antibodies with higher affinity constants for the hormone must be used.

Furthermore, a monoclonal antibody produced using cell fusion technology is used to recognize a common subunit of a physiologically active substance, which is composed of two subunits that cross-react with the substance to be measured, as the antibody used for measurement. By treating with monoclonal antibodies with different sites and binding the common subunit part of the intersecting physiologically active substances to the monoclonal antibody in advance and blocking,
It is thought that it is also possible to eliminate the influence on the measurement by preventing the antibody that recognizes the common subunit used from binding to the cross-reacting physiologically active substance.

However, when this method is applied to the one-step sandwich method, it often has the disadvantage that the monoclonal antibody used for blocking also binds to the substance to be measured, and the antibody against the common subunit used for measurement does not bind. be. Therefore, there is a need to establish a one-step sandwich method for biologically active substances consisting of two subunits that does not have these drawbacks.

(Means for Solving the Problems) The present inventor has arrived at the present invention as a result of intensive research. That is, the present invention combines an antibody against one subunit of a physiologically active substance consisting of two subunits to an insoluble substance to form an insoluble antibody, and an antibody against the other subunit to form a labeled antibody by labeling it with an enzyme. A physiologically active substance composed of subunits is reacted simultaneously with an insoluble antibody and an enzyme-labeled antibody, and the enzyme activity of the enzyme-labeled antibody bound to the insoluble antibody via the physiologically active substance composed of two subunits or the enzyme label that is not bound is determined. In enzyme immunoassay that measures the concentration of a physiologically active substance consisting of two subunits by measuring the enzyme activity of an antibody, a subunit that is common to one of the subunits of the physiologically active substance consisting of the two subunits. A monoclonal antibody that recognizes the bonded cross-linked portion of two subunits of another physiologically active substance having the above properties is allowed to coexist with the sample, and the physiologically active substance composed of the two subunits is specifically measured. This is an enzyme-linked immunosorbent assay.

In the present invention, physiologically active substances consisting of two subunits include, for example, thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle stimulating hormone (FSH).
, chorionic gonadotropin (HCG), and other hormones. The two subunits in these physiologically active substances are the α subunit and the β subunit.

A physiologically active substance consisting of two subunits and another physiologically active substance having a subunit in common with one of the subunits include, for example, a TS having a common α subunit.
Examples include H and HCG. Other physiologically active substances in the present invention also have two subunits.

In the present invention, when carrying out the one-step sandwich method using antibodies against each subunit of a physiologically active substance consisting of two subunits, it is possible to detect other physiologically active substances that are common to one of the constituent subunits. This effect is avoided by the presence of a monoclonal antibody that recognizes the binding portion of the two subunits of the active substance, the so-called cross-reacting substance, the α subunit and the β subunit. Specifically, an antibody that recognizes one subunit, such as the α subunit, or an antibody that recognizes another subunit, such as the β subunit, is bound to a solid phase, and an antibody that recognizes a subunit other than the solid phase is used. to mark. When measuring a physiologically active substance to be measured, first check the affinity constant or the degree of cross-reactivity for other physiologically active substances that cross-react with antibodies that recognize common subunits, and then We will investigate whether there are any bioactive substances that have a high degree of cross-reactivity or that have a large degree of cross-reactivity. When the existence of such a physiologically active substance is recognized, a monoclonal antibody that recognizes the binding portion of the two cross-reacting subunits of the physiologically active substance, namely the α subunit and the β subunit, is made to coexist. By doing so, the monoclonal antibody selectively binds to the cross-reacting physiologically active substance, and the binding of the labeled antibody is sterically inhibited so that it does not affect the target physiologically active substance measurement system.

More specifically, in the case of a hormone consisting of two subunits, in the case of TSH measurement, an antibody against the β subunit of TSH is used as the solid phase, and an antibody against the α subunit of TSH is used as the labeled antibody. When the cross-over ratio of antibodies against the α subunit of TSH to other hormones is 800100% and TSH 27.8%, the labeled antibody that recognizes the α subunit subjected to measurement binds more to HCG, and TSH It is thought that the bond with Therefore, by adding a monoclonal antibody that recognizes the binding portion between the α subunit and β subunit of HCG, the influence of high concentrations of HCG can be avoided during TSH measurement.

Furthermore, a monoclonal antibody that recognizes the binding portion of two subunits of a physiologically active substance that cross-reacts with the target antibody of the physiologically active substance is produced according to a conventional method. The amount added is not particularly limited. This monoclonal antibody can be produced using common cell fusion techniques. That is,
Mice are immunized with a biologically active substance consisting of two subunits that cause cross-reactivity together with an adjuvant, and the obtained lung cells and mouse myeloma cells are fused using a cell fusion agent such as polyethylene glycol, and after culturing in HAT medium. Monoclonal antibodies can be obtained by finding and culturing hybridomas among the surviving fused cells that do not react with each subunit but recognize only the physiologically active substance made up of two subunits.

In addition, as antibodies against physiologically active substances used for measurement, animals are immunized with each subunit of a physiologically active substance consisting of two subunits according to the method for obtaining general antiserum, and additional antibodies are added if necessary. It can be obtained by immunization. Furthermore, monoclonal antibodies obtained by cell fusion technology can also be used, but from the viewpoint of specificity, it is preferable to use monoclonal antibodies.

The solid phase to which the antibody against the physiologically active substance used for measurement is bound is generally insoluble in water and may be any solid phase as long as it is easy to handle, but particularly preferred are polystyrene balls, polystyrene tubes, and glass. Peas,
A piece of silicone or the like is suitable.

Enzymes used for labeling may be of any origin, but inexpensive and easily available enzymes include β-galactosidase, alkaline phosphatase, peroxidase, glucose oxidase, and the like. Known labeling techniques may be used to label antibodies with these enzymes. For example, cross-linking may be performed using well-known bifunctional reagents such as glutaraldehyde or maleimide, or if the enzyme has a sugar chain in its molecule, such as peroxidase, the sugar chain may be linked with periodic acid. It may be oxidized with acid to expose the aldehyde group and then bonded to the amine group of the antibody. The labeled antibody thus obtained is
It is advantageous to use a fraction that has high enzyme activity and antibody activity by performing appropriate purification operations such as dialysis and gel filtration.

In the present invention, an insoluble antibody, an enzyme-labeled antibody, and a sample are reacted in the presence of a monoclonal antibody that recognizes the bonded crosslinked portion of two subunits of a physiologically active substance with the above-mentioned ability, and an enzyme-labeled antibody bound to the insoluble antibody is used. By measuring the enzyme activity of the antibody or the enzyme activity of the unbound enzyme-labeled antibody, the concentration of the target physiologically active substance consisting of two subunits is determined.

(Example) Next, the present invention will be specifically described using Examples, but the present invention is not limited to the Examples.

In the case of TSH measurement, a monoclonal antibody (manufactured by Cambridge, USA) that specifically reacts with the TSH β subunit was used as a control solid-phase bound antibody in phosphate buffered saline (pH 7.2, 0.01
M) to a concentration of 10 ug/- and bonded to a 1/4 inch polystyrene ball by physical adsorption. The cross-reactivity of a monoclonal antibody against the α subunit (manufactured by Cambridge, USA) as a labeled antibody with other hormones is as follows.

HCG: 100% TSH: 27.8% LH: 33.7% FSH: 5.4% These monoclonal antibodies against the α subunit were oxidized using peroxidase (Toyobo type, grade I- C) was labeled and purified using Sephadec G-200 to obtain a labeled antibody. As standard TSH, human TSH from Calbiochem, USA was used, and 1
%BSA in phosphate buffered saline, water (pH 7.2,
0.01M) to the specified amount, and diluted with standard TS
It was set as H. In a test tube, put 100μ of this standard TSH, 500μ of the enzyme-labeled antibody diluted in advance at a dilution ratio that would give an appropriate absorbance in a preliminary test, and one antibody-bound ball, incubate at 37°C for 2 hours, and then add distilled water. 3
After washing twice, the balls were transferred to a 300 μm citric acid-phosphate buffered coloring solution containing 0.02% hydrogen peroxide and 3 mg/d concentration of orthophenylene diamine, and enzymatic reaction was performed for 30 minutes, followed by 1 d of 1N sulfuric acid. The reaction was stopped, and the absorbance of 492nI11 was determined using a photometer. When HCG is not present in the substance to be measured, it is measured correctly, but when there is no coexisting HCG, it is measured correctly, but when the coexisting HCG is 10,000 to 50, which is the level of pregnant women.
,000 m1/-, the measured value of TSH showed a low value as shown in Table 1.

Example 1 HCG was added in the measurement system of the control example, that is, in the enzyme-labeled antibody solution.
A monoclonal antibody (manufactured by Cambridge, USA) that recognizes the binding site between the α and β subunits of
A concentration of 0 Mg/- was present.

Absorbance was determined in the same manner as the control example. The results are shown in Table 1. Pregnant women's level of 10.000 to 5 in the measurement control substance
It is clear from Table 1 that even if 0.000+alU/- of HCG is present, the effect is almost eliminated.

Table I Absorbance equivalent to TSH 10 μU/- Control example for LH measurement As a solid phase-bound antibody, a monoclonal antibody (manufactured by Cambridge, USA) that specifically reacts with the LHβ subunit was added to phosphate buffered saline (pH 7, 2). ,0,01M
) to a concentration of 10 Mg/- and bonded to a 1/4 inch polystyrene ball by physical adsorption. The cross-reactivity of a monoclonal antibody against the α subunit (manufactured by Cambridge, USA) as a labeled antibody with other hormones is as follows.

HCG: 100% TSH: 85.0% LH: 90.0% FSH: 1B. -C) was labeled and used as a labeled antibody. As standard LH, human LH from Calbiochem, USA was used and diluted to a predetermined amount with phosphate buffered saline (pH 7, 2°, 0.01M) containing 1% BSA. This standard LH1 in a test tube
00μ, an enzyme-labeled antibody diluted in a preliminary test to a dilution ratio of 500μ to obtain an appropriate absorbance, and one antibody-bound ball, incubated at 37°C for 2 hours, and washed 3 times with distilled water. Hydrogen peroxide 0.02%, 3
Transfer the ball to 300μ of a citric acid-phosphate buffered coloring solution containing orthophenylenediamine at +ig/dtlA degrees, allow the enzymatic reaction to occur for 30 minutes, stop the reaction with 1N sulfuric acid, and measure the color using a photometer at 492nm. Find the absorbance of If HCG is not present in the substance to be measured, it will be measured correctly, but if HcG is not present in pregnant women/170) 10
,000~50,000m1U/-, L
The measured value of H shows a low value as shown in Table 2.

Example 2 HOG was added in the measurement system of the control example, that is, in the enzyme-labeled antibody solution.
A monoclonal antibody (manufactured by Cambridge, USA) that recognizes the binding site between the α and β subunits of
0 MgldtllA was present.

Absorbance was determined in the same manner as the control example. The results are shown in Table 2. Pregnant women's level of 10.000 to 5 in the measurement control substance
It is clear from Table 2 that even if 0,000 m1U/11j of HCG is present, the effect is almost eliminated.

Table 2 Absorbance 4 equivalent to LH 50 mU/d Effect of the invention Conventionally, a two-step sandwich method was used to measure trace components in body fluids using antigen-antibody reactions for physiologically active substances consisting of two subunits. This method was the most popular, but it had drawbacks such as complicated operations and long reaction times. By using the present invention, even if there are hormones that share the same subunit coexisting in the measurement target, measurements can be made easily and in a short time without being affected by the presence of hormones. It becomes possible.

Claims (1)

[Claims] An antibody against one subunit of a physiologically active substance consisting of two subunits is bound to an insoluble substance to form an insoluble antibody, and an antibody against the other subunit is labeled with an enzyme to form a labeled antibody. The active substance is reacted simultaneously with the insoluble antibody and the enzyme-labeled antibody, and the enzymatic activity of the enzyme-labeled antibody bound to the insoluble antibody or the enzyme activity of the enzyme-labeled antibody that is not bound to the insoluble antibody is determined by reacting the active substance simultaneously with the insoluble antibody and the enzyme-labeled antibody. In an enzyme immunoassay method that measures the concentration of a physiologically active substance composed of two subunits, the concentration of a physiologically active substance composed of two subunits is determined by measuring the concentration of another physiological substance having a common subunit with one of the subunits of the physiologically active substance composed of two subunits. An enzyme immunoassay method characterized in that a monoclonal antibody that recognizes the bonded crosslinked portion of two subunits of an active substance is allowed to coexist with a sample, and a physiologically active substance composed of the two subunits is specifically measured. .