JP3418429B2 - Free ligand assay - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring a free ligand in a sample, and more particularly to a method for specifically measuring a free ligand in the presence of a binding equilibrium between the ligand and a binding substance. It is a thing.

[0002]

2. Description of the Related Art In a living body, a physiologically active substance may coexist with a specific binding substance, and a binding equilibrium may exist between them. At this time, since only the released physiologically active substance has the original physiological activity, the effective amount of the physiologically active substance is affected by the affinity between the physiologically active substance and the binding substance and the amount of the binding substance. . Methods such as extraction [Daughaday,
WH et al .: J. Clin. Endocrinol. Metab., 51: 781, 198
[0] is used to dissociate the physiologically active substance and its binding substance to measure the total amount of the physiologically active substance, the amount of the effective physiologically active substance alone cannot be measured. A method of measuring the amount of is desired.

Conventionally, several methods have been attempted as means for measuring only fL in a mixture of free ligand (fL) and bound ligand (LB). One is to separate the two by using the difference in the physical properties of fL and LB. Information on the fL concentration in the mixture can be obtained by measuring L using the fL fraction obtained by separating LB and B from the mixture as a sample. For example,
Equilibrium dialysis method [Sterling, K. et al .: J. Clin. Invest., 45: 1
53, 1966] and ultrafiltration [Schussler, GC et al .: J. C.
lin. Endocrinol. Metab., 27: 242, 1967] uses a membrane that allows only fL to pass through.

When only fL is separated from the mixture of fL and LB, the time required for separation becomes a problem in any method for the reasons described below. That is, in general,
In the mixture of fL and LB, both are in a dynamic equilibrium state (fL +
B ⇔ LB). In this state, when an attempt is made to separate fL and LB using, for example, an equilibrium dialysis method, the equilibrium moves to the left as fL diffuses from the test sample in the dialysis membrane into the extramembrane fluid, and fL is replenished. The longer the time required for separation, the more fL
Will increase. As a result, the measured value of L contains a positive error. On the contrary, in the ultrafiltration method, since the equilibrium moves to the right due to concentration, it is considered that the concentration of fL contained in the filtrate decreases with time. In addition, it is impossible to use this method when the molecular weights of B and L have no difference.

In the separation method using the above membrane, the time required for separation is proportional to the molecular weight of L. That is, the larger the molecular weight, the lower the rate of permeation through the membrane, and the longer the time required for separation. Since the error included in the measured value of L also becomes large, fL that can be measured by the separation method using the above-mentioned membrane is limited.

Another method for separating both fL and LB using the difference in physical properties is to use gel filtration [Zapf, J. et al .: J. Clin. Invest., 77: 1768, 198].
6] or a method of separating by utilizing the difference in hydrophobicity [Hizuka,
N. et al .: Growth Regulation, 1:51, 1991] has been tried. Even in these methods, the time required for separation influences the result. Further, in all the above methods, the operation up to the measurement is complicated.

Another method for measuring only fL in a mixture of fL and LB is to add a binding substance (C) that reacts only with fL, such as an antibody, to the mixture of fL and LB, and To capture. As a method for measuring L, there are a competitive method using labeled L or a substance (L ′) similar to L, and a sandwich method using a labeled second binding substance. In order to maximize the effective use of C added to the test sample, the reaction between fL and C is carried out until equilibrium is reached, but when C is added in the state where a binding equilibrium exists between L and B, A competition is established between C, and a part of L, which was in a bound state with B before the start of the reaction, is bound with C (LB + C⇔LC + B).

In order to eliminate the influence of the competition between B and C on the measured value of fL, a method of adding a binding substance (C ') having a low affinity for L may be adopted. In this case, the reaction between fL and C ′ is slower than that when C, which has a high affinity for L, is used.
The binding equilibrium between L and B shifts before L is captured, and an amount of L that supplements the captured fL is released from LB.

A problem with the method for measuring fL in a test sample using C or C'is that the measured value tends to be higher than the true value and that the reproducibility is poor. Is mentioned. Since the affinity and the amount ratio of B and C or C'to L have a great influence on the measured value, a slight difference in the conditions during the measurement causes an error in the measured value. For example, it is generally known that the affinity of a binding substance such as a binding protein or antibody for a ligand greatly changes depending on temperature, pH, and a coexisting substance. It is easily inferred that the change in affinity affects the reaction for measurement. In the competitive method, since labeled L or L'is added,
The measured value will be further influenced by various substances.

The hypothesis that the rate at which L and C bind to each other is not limited by the rate at which L dissociates from LB is disclosed in Japanese Patent Laid-Open No.
Although it is found in Japanese Patent Laid-Open No. 5-146043, there is no mention of a measure for eliminating the influence of fL caused by the shift of the equilibrium.

Further, Japanese Patent Publication No. 62-43138 discloses a quantitative method for determining the amount of an immunologically active substance from the rate of immunological reaction. However, in this method, the measurement using the latex agglutination reaction is taken as an example, the absorbance at two different time points is measured, and the amount of the immunologically active substance is calculated from the increase in the absorbance,
It is only mentioned that an easy and quick measurement is possible.

[0012]

DISCLOSURE OF THE INVENTION The present invention provides a ligand (L) to be measured and a substance (B) having a binding property to L.
When there is a binding equilibrium between the two and the measurement system of L influences the equilibrium, it provides a means for easily knowing the amount of free ligand (fL) before the equilibrium is affected.

[0013]

In this method, the reaction for measurement is carried out for a sufficient time for the reaction to reach equilibrium, so that the measurement of fL originally present in the test sample is carried out. The fL in the mixture of L and B is simply and rapidly measured based on the finding that the increase in the reaction product at the start of the reaction reflects only the fL concentration after considering the effect on the Is the way.

That is, according to the present invention, even when the L measuring system influences the equilibrium, only the fL existing before the equilibrium is transferred is involved in the reaction immediately after the reaction for measurement is started. This is what you do. L
Using a measurement system for measuring L by adding a substance (C) having a binding property to (C) to an insoluble solid in a state of being immobilized on an insoluble solid, the equilibrium resulting from the influence of the measurement system on the equilibrium Is a method for measuring fL that was initially present in a test sample, characterized in that the reaction was terminated in the state before the movement of caused a significant error in the measured value of L. According to the method of the present invention, the concentration of fL originally present in the mixture of L and B in a dynamic equilibrium state can be measured more accurately and easily.

As L in the present invention, any substance in which a substance having a binding property with L exists is applicable.
For example, hormones (growth hormone, adrenocorticotropic hormone secretory factor, thyroxine, triiodothyronine, insulin), insulin-like growth factor I (IGF-I),
Examples include all kinds of antigenic substances. INDUSTRIAL APPLICABILITY According to the present invention, IGF having a large molecular weight is difficult to be fractionated by a membrane.
It is particularly effective in measuring −I and the like. Examples of the substance (B) capable of binding to L in the test sample include a specific binding protein, a receptor, and a specific antibody. In the case of the above-mentioned IGF-I, IGFBP-1, IGF as the main binding protein present in blood
BP-3 can be mentioned [Naomi Kozuka: Hormone and Clinical, 3
9: 1261, 1991].

When a substance (C) having a binding property to L is added to measure L, C may be the same as or different from B, but a test sample is preferred. It should be added in a way that does not change the liquid properties of. In an embodiment of the method of the invention, a mouse monoclonal antibody (primary antibody) that specifically binds to IGF-I.
Was bound to a plastic bead solid phase and subjected to measurement.

When measuring fL, it is desirable to use the sandwich method, that is, the second binding substance (D) for L.
The method of using should be adopted. D is required to be able to be bonded to L in a state of being bonded to C. Also in the method of the present invention, IGF
A mouse monoclonal antibody (second antibody) capable of sandwiching I was adopted.

The reaction for measuring fL includes a reaction (first reaction) in which L is captured by C immobilized on an insoluble solid and a C reaction.
The most preferable method for measuring fL using this method is to carry out the reaction (second reaction) in which L immobilized to an insoluble solid is bound with labeled D via a second step. In the embodiment of the method of the present invention, the test sample is reacted with the first antibody-immobilized beads and unreacted IGF-I is removed by washing. The ¹²⁵ I-labeled secondary antibody is further reacted to remove unreacted secondary antibody, and the radioactivity remaining on the beads is measured.

All of the above points of concern are means for subjecting the test sample to the measurement without dilution. That is, in order to realize accurate measurement of fL, it is necessary to provide the measurement without disturbing the equilibrium between L and B when the test sample is collected. Of course, the reaction for measurement must be performed under sufficient temperature control.

In the present invention, the most important point in terminating the reaction before being affected by L supplied from LB is the reaction time of the reaction corresponding to the first reaction when the above sandwich method is used. Is the prescribed method. That is, in order to measure fL that was initially present in the test sample,
The point is to stop the reaction for trapping L before producing a significant error. Taking the above sandwich method as an example, immediately after adding C immobilized on an insoluble solid to a test sample, fL
Begins to bond with C. When fL and C bind and the amount of fL in the test sample decreases, the binding equilibrium between L and B (L + B⇔
LB) affected but fL used for measurement
(Ie, the amount of fL bound to C) is sufficiently small,
The reaction rate of fL and C depends on the concentrations of fL and C.
When the amount of C added to the test sample is constant, the reaction rate depends on the total fL amount. In order to quantify the amount of fL in the test sample using this method, it is necessary to prepare a calibration curve by simultaneously subjecting a standard sample containing a known concentration of fL to the measurement.

The optimum reaction time in the method of the present invention refers to a period in which the reaction rates of fL and C in the standard sample and the test sample equally reflect the initial fL concentration.
During this period, the signals of the standard sample and the test sample increased in proportion to the reaction time. By preparing a calibration curve based on the signal obtained from the standard sample and fitting the signal obtained from the test sample to the calibration curve, the fL concentration initially present in the test sample can be determined. Within the period in which the reaction rates of fL and C in the standard sample and the test sample equally reflect the initial fL concentration, regardless of the reaction time (that is, regardless of the magnitude of the signal), the calibration The reading of fL concentration initially present in the test sample determined using the line is constant. In carrying out the method of the present invention, the most important thing is to determine the time from when the reaction for measurement is started until the reading of the fL concentration initially present in the test sample starts to change. is there. The time within the range where the fL concentration reading initially present in the test sample does not change is set as the optimum reaction time in the method of the present invention. The factor that defines the optimum reaction time in the present invention is the amount of C added. The condition is that the amount of C to be added is constant, but the amount of C to be added must be determined in consideration of the calibration range necessary for measurement. That is, the larger the amount of C added, the faster the reaction for measurement. As a result, the optimum reaction time in the present invention is shortened. When the amount of C added is small, the calibration range becomes narrow. It is necessary to set the amount of C to be added so that the optimum reaction time in the present invention is long enough to allow accurate measurement and the calibration range is sufficient for measurement.
Since the amount of C satisfying the above conditions is affected by the amount ratio of L to B and C and the affinity, it is desirable to determine for each measurement system.

[0022]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Example Establishment of assay method for free insulin-like growth factor I (fIGF-I) a) Preparation of IGF-I antibody beads From spleen cells and mouse myeloma cells obtained by immunizing mice with IGF-I, Tomonoyama Sakuji, Ando Tamie ed., “Monoclonal Antibody Experiment Manual” (1987) Kodansha, the monoclonal hybridoma obtained by the method described in
Anti-IGF-I monoclonal antibody was prepared by purifying ascites. The prepared anti-IGF-I monoclonal antibody was physically adsorbed on polystyrene beads under alkaline conditions and subjected to measurement.

B) Preparation of iodinated IGF-I antibody ( ¹²⁵ I) Iodine ¹²⁵ was introduced by the chloramine T method into the monoclonal antibody against IGF-I obtained in the same manner as in the above a). The iodinated IGF-I antibody ( ¹²⁵ I) was prepared using the monoclonal antibody used in a) above and a monoclonal antibody capable of forming a sandwich against IGF-I.

C) Preparation of standard IGF-I Human IGF-I obtained from Toyobo Co., Ltd. was added to a phosphate buffer containing bovine serum albumin at 0.1 to 10 ng / m.
It was prepared to be l.

D) Obtaining Human Plasma Sample Blood was collected from a healthy person using an EDTA blood collection tube, and after plasma separation, it was immediately frozen and stored until use.

E) Measurement of the effect of the first reaction time on the binding rate. Standard IGF-I, human plasma sample and beads were each added to 3
Incubate at 7 ° C for 30 minutes (preliminary incubation). After that, the beads are rapidly added to the standard IGF-I and the human plasma sample, and further incubated at 37 ° C (first reaction). The time of the first reaction was changed to see the effect on the bound radioactivity. After removing the reaction solution, the beads after the first reaction are washed with purified water,
Phosphate buffer containing iodinated IGF-I antibody ( ¹²⁵ I) was added. This was shaken at room temperature (second reaction), the reaction solution was removed and washed, and then the radioactivity remaining on the beads was measured.
In the second reaction, the time necessary and sufficient for obtaining the maximum binding rate was set. That is, the second reaction time was set to 3 hours from the result of the preliminary experiment. The first reaction time was plotted on the horizontal axis and the bound radioactivity (cpm) was plotted on the vertical axis, and the measured values of each standard IGF-I and human plasma specimen IGF-I were plotted (FIG. 1). Immediately after starting the reaction, the bound radioactivity increased as the reaction time increased for both the standard IGF-I and the human plasma sample (period in Fig. 1-a), but eventually the standard I
Differences were observed in the changes in the bound radioactivity between GF-I and human plasma specimens (period in Fig. 1-b), and human plasma was tested even after the maximum bound radioactivity of standard IGF-I was reached. The bound radioactivity of the specimen continued to increase gradually (period in Figure 1-b).

F) Effect of first reaction time on analyte reading From the bound radioactivity of the standard IGF-I obtained in d) above,
A calibration curve was prepared at each reaction time (Fig. 2). The bound radioactivity of the human plasma sample at the same reaction time was inserted into the calibration curve to determine the sample IGF-I concentration. The horizontal axis represents the first reaction time, and the vertical axis represents the sample IGF-I concentration reading (FIG. 3). Sample IG within the period corresponding to the period in Fig. 1-a
It can be seen that the FI density reading is constant.

G) Determination of optimal first reaction time In order to improve the measurement accuracy, the first reaction time is the sample IGF-
It is desirable to set a long value within a range where the I density reading does not change. In the examples of the method of the present invention, 5 minutes was used as the reaction time of the first reaction.

H) Measurement of fL concentration initially present in human plasma samples Free IGF-I (fI) optimized as in the above examples
The GF-I) assay was used to determine the fL concentration initially present in the human plasma sample, which was 2.1 ng / ml. For confirmation, the measurement was carried out by changing the first reaction time to 0.5 minutes, 1 minute, 3 minutes, 5 minutes, 10 minutes, and all were 2.
It was 1 to 2.3 ng / ml, and the reproducibility was good.

Comparative Example fIGF-I measurement using gel filtration method a) Preparation of gel filtration column A gel filtration column was prepared using Sephadex G-75 manufactured by Pharmacia.

B) Confirmation of elution fraction of fIGF-I IGF-I ( ¹²⁵ I) iodide was applied to a gel filtration column,
The elution fraction of fIGF-I was confirmed using the radioactivity of the eluate as an index (Fig. 4-a).

C) Confirmation of Elution Fraction of Bound IGF-I IGF-I ( ¹²⁵ I) iodide was stored overnight at 4 ° C. together with the human plasma sample obtained in Example d). Since human plasma has an excess amount of binding protein as compared with IGF-I, most of iodoiodinated IGF-I ( ¹²⁵ I) binds to the binding protein. The elution fraction of bound IGF-I was confirmed by gel filtration of this sample (FIG. 4-b). It was confirmed from FIGS. 4A and 4B that fIGF-I and bound IGF-I could be separated.

D) Gel filtration of human plasma sample The human plasma sample used in the above Example h) was applied to a gel filtration column, and the fIGF-I elution fraction confirmed in the above Comparative Example b) was collected. The collected fIGF-I elution fractions were lyophilized and stored until they were used for measurement.

E) Measurement of elution fraction of fIGF-I in human plasma sample According to the fIGF-I measuring method established in the above Example, fI in human plasma sample obtained in the above Comparative Example d).
The IGF-I content of the GF-I eluted fraction was measured. The bound IGF was added to the elution fraction of fIGF-I according to Comparative Example c).
Since it was confirmed that -I was not mixed, the reaction time of the first reaction was changed so that the maximum binding rate was obtained.

F) Results fIGF- in human plasma sample determined from the IGF-I content in the elution fraction of fIGF-I by gel filtration of human plasma sample
The I concentration was 2.0 ng / ml, supporting the above example.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the time of the first reaction and the bound radioactivity.

FIG. 2 is a standard IGF-I calibration curve obtained from each first reaction time.

FIG. 3 is a diagram showing the influence of the time of the first reaction on the sample IGF-I concentration reading.

FIG. 4 is a diagram showing an elution pattern of free (a) and bound (b) IGF-I on a gel filtration column.

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Claims (5)

(57) [Claims] 1. A test sample in the case where a test substance (L) which is a ligand and a protein (B) capable of binding to L coexist in the test sample and a binding equilibrium exists between L and B. A method for measuring fL for specifically measuring only the free ligand (fL) initially present in the medium, wherein a substance (C) having a binding property to L is immobilized on an insoluble carrier. A measurement method characterized by terminating the reaction for measurement before being affected by L supplied from a bound ligand (LB) by adding it to a test sample. 2. The assay method according to claim 1, wherein B is an antibody against L, a receptor or a binding protein. 3. The assay method according to claim 1, wherein C is an antibody against L, a receptor or a binding protein. 4. The measuring method according to claim 1, wherein C is immobilized on an inorganic substance, plastic or organic polymer gel. 5. The measurement method according to claim 1, wherein the measurement target fL is insulin-like growth factor I.