JPH0458155A - Bf separation for immunological measurement - Google Patents

Abstract

PURPOSE:To provide the method effective in terms of production, management or speeding up of measurement without having the possibility of deactivation upon lapse of time by previously conjugating biotin with a solid phase for BF sepn. and conjugating an antigen labeled with the biotin, etc. via adipin with this solid phase at the time of measurement. CONSTITUTION:The biotin B is previously conjugated with the solid phase 1 and antibody 3 labeled with the biotin B is conjugated via the adipin A therewith. The antigen 2 labeled with the biotin B is conjugated via the adipin A with the biotin B conjugated with the solid phase 1. The biotin B is an org. compd. and is hardly decomposed, like protein, etc., by the influence of temp., enzyme, etc., and is relatively stable. The biotin-labeled antigen2 or antibody 3 is conjugated via the adipin A with the biotin directly conjugated with the solid phase 1 and, therefore, the solid phase 1 to be prepd. is the common solid phase conjugated with the biotin B. The production line is thereby simplified and the cost of production is reduced. In addition, the management is facilitated.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to BF separation for immunoassays used when measuring each substance a in human or animal body fluids using antigen-antibody reactions. Regarding the law.

(Conventional techniques and inventions! &i) Hormones present in body fluids such as blood and urine, substances derived from cancer cells, substances derived from pathogenic microorganisms, autoantibodies, or pathogenic microorganisms When detecting and measuring antibodies, etc.
An antigen-antibody reaction in which an antibody binds to an antigen is used. This measurement method using antigen-antibody reaction (hereinafter abbreviated as immunoassay) requires BF separation (B is antibody or antigen bound to antigen or antibody, F is unbound free antigen or antibody) as.

(1) Enzyme immunoassay (Enzywie fwunoa)
ssay-E IA) (2) Radioimmunoassay
say-RIA) (3) Fluorescence immunoassay (Fluoro Immunoassay)
(4) Time-resolved fluorescence immunoassay (Time Re5ol)
vedFluoro Immunoassay-TR
-FIA) etc.

As shown in FIG. 9 for the sandwich EIA method, the enzyme immunoassay method uses a solid phase 1 (this solid phase is a container-shaped plate as shown in FIG. 1, or a bead-shaped solid phase). An antibody (which may be an antigen) 3 corresponding to the antigen (which may be an antibody) 2 to be measured is bound in advance to the surface of a tube-shaped object or a tube-shaped object. When antibody 3A labeled with enzyme E is added, a side-ditch-like antigen-antibody complex consisting of antibody 3-antigen 2-antibody 3A as shown in the figure is formed in solution 4, and then solution 4 is washed away and antigen 2 is added. This method uses a colorimetric method to measure the amount of enzyme E that corresponds to the amount of . That is, considering that the amount of antigen 2 that reacts with antibody 3A bound to solid phase 1 is proportional to the content of antigen 2 in liquid 4,
This method measures the amount of antigen 2 as the amount of enzyme E. In addition, to measure the intensity of enzyme E, after draining liquid 4 and washing solid phase 1, a liquid containing a substance (substrate) that is colored or decolored by enzyme E is added to the plate, and the change in absorbance is measured. Do it by doing this.

The radioimmunoassay method uses a radioactive element as a labeling substance instead of the enzyme E, and the amount of antigen is measured from the amount of radioactive element after the liquid has flowed out.The fluorescence immunoassay method uses an enzyme E as a labeling substance. In this method, a fluorescent substance is used instead of E, and the amount of antigen is measured from the amount of light from the fluorescent substance after the liquid has flowed out.

This conventional measurement method has the following problems: (1) Since the antibody (or antigen) 3 is bound to the solid phase in advance, there may be cases where the antibody (or antigen) 3 is unstable. To,
There may be problems with the inactivation of antibodies and antigens over time, or with stability during storage.

(2) A solid phase 1 bound with antibodies (or anti-FK) 3 corresponding to each antigen (or antibody) 2 to be measured must be prepared in advance, and it differs for each of the hundreds of types of antigens or antibodies. Preparing the solid phase 1 is complicated in terms of production or management, and also leads to an increase in production costs.

(3) Microscopically, unless the antigen (or antibody) 2 in the liquid 4 comes close to the antibody (or antigen) 3 bound to the solid phase 1, an antigen-antibody reaction will not occur, so to speak, a solid phase reaction. Therefore, the reaction rate is slow and measurement takes time.

As a method to solve the above problems (2) or (3), the BF separation method using atomine and biotin [Japanese Society of Obstetrics and Gynecology ACTA 0BST GYNAECJP]
N Vol, 3[i No, 5 P763~P7
70]. As shown in Figure 810, avidin A is bound to the solid phase 1, and biotin B-labeled antigen 2 and enzyme E-labeled antibody 3 are reacted in advance in a separate test tube. This is a separation method that utilizes the fact that when the solution is added to the solution, avidin A and biotin B bind together, and the complex of biotin B-labeled antibody 2 and enzyme-labeled antibody 3 binds to a solid phase. A method in which this method is applied to the sandwich RIA method and beads are used as the solid phase has also been reported [Nuclear Medicine: Vol. 27, No. 2 (1990) P1
55-P16:l.

As described above, according to the method of binding adipine A to the solid phase 1, there are problems with deactivation of avidin A over time or stability during storage.

In view of the above-mentioned problems, an object of the present invention is to provide a BF separation method that is free from the risk of deactivation over time and is advantageous in terms of speeding up production, management, and measurement.

(Means for Solving the Problems) In order to achieve the above object, the BF separation method according to the present invention has the following features:
Biotin (including its analogs and derivatives) is bound to the solid phase for BF separation in advance, and biotin (including its analogs and derivatives) is bonded to the solid phase via avidin (including its analogs) during measurement. It is characterized by binding an antigen or antibody labeled with (including).

FIG. 1 is a diagram explaining the measurement principle of the present invention, in which biotin B is bound to a solid phase material in advance, and biotin B
Antibody 3 labeled with (2 is an antigen forming a complex with antibody 3) is bound via avidin A. In addition, as shown in Fig. 2, antigen 2 labeled with biotin B (3 is an antibody forming a complex with antigen 2) is added to biotin B bound to solid phase 1 via avidin A. Join.

Note that similar substances or derivatives thereof can be used instead of biotin and avidin, respectively. Specifically, for example, instead of biotin, its derivative 2-
Imino-biotin (2-1m1n-obiotin)
, biocytin, bisnorbiotin, tetranorbiotin (
tet-oranorbiotin), desthiobiotin, oxybiotin (o
A biotin-related substance such as xybiotin) can be used, and as an avidin analogue, for example, streptavidin (a substance similar to avidin obtained from actinomycetes) can be used.

(Action) Avidin has 4 binding sites with biotin,
Taking advantage of this, the present invention binds biotin to a solid phase in advance, and binds an antigen or antibody labeled with biotin or the like to this biotin via avidin. Biotin is a stable substance and rarely loses its activity over time.

Furthermore, avidin has extremely high affinity with biotin (dissociation constant = 10-I5M), and forms a complex with extremely stronger binding force than antigen-antibody. Furthermore, in the present invention, the antigen-antibody reaction occurs in a liquid phase.

(Example 1) Figure 3 is a diagram explaining the measurement principle when the present invention is applied to [sandwich EIA], in which avidin A, anti-jK2, and biotin are added to the solid phase (well of the plate) to which biotin B is bound. Add B-labeled antibody 3 and enzyme E-labeled antibody 3A,
After washing, measure the amount of enzyme remaining on the solid phase surface. That is,
Of the two antibodies 3.3A that sandwich antigen 2 and bind to it, one antibody 3 is labeled with biotin B, and the biotin B of this biotin-labeled antibody 3 is immobilized via avidin A. Biotin B pre-bound to phase I
The results of a specific experiment of this Example 1, which is a method of immobilizing a complex of antigen 2 and antibody 3A on a solid phase by binding to a solid phase, will be described regarding the measurement of human serum albumin. Add 200 g/ml of biotin-labeled bovine serum albumin to the wells of the EIA plate.
After adding g+ and reacting at room temperature for 2 hours to bind biotin-labeled bovine serum albumin, the plate was washed with a washing solution to prepare a biotin-bound plate. Subsequently, the following reagents were added and reacted at room temperature for 2 hours.

Reagent 1 (antigen): Human serum albumin 50JL1 (0-
1000 ng/ml) Reagent 2 (biotin-labeled antibody): biotin-labeled anti-human serum albumin antibody 50
JL+ (20 g/■1) Reagent 3 (enzyme-labeled antibody): Veroxidase-labeled anti-human serum albumin antibody 50 I
Ll (5u-g/kg) Reagent 4 (avidin): Avidin 50g+(10ILg/■1) After the reaction, add peroxidase substrate solution 2 0.251g/
Seal I OJ Retophenylenecyamine・2HCl-0,01
5%H2020, 11MNaz HPO, -0,044
After adding 200 ILl of M citric acid (PH5,4) and reacting at room temperature for 30 minutes, the absorbance at a wavelength of 492 nm was measured to prepare a calibration curve. Method 1 in FIG. 4 is its calibration curve, and a clear correlation between albumin concentration and absorbance was obtained, confirming that this method can be used.

Further, FIG. 4 shows a comparison of calibration curves obtained by changing the procedure for adding the four types of reagents. In the fJS4 diagram, methods 1 to 4 each involve adding reagents in the following steps.

Method 1: Add reagents 1-4 simultaneously as described above.

Method 2: Add reagents 1 to 3 and react for 2 hours, then add reagent 4 and react for 1 hour. Method 3. Add reagent 4 and react for 1 hour, then add reagents 1 to 3 and react for 2 hours. Method 4: Add reagents 1 and 3.4 and react for 2 hours, then add reagent 2 and react for 1 hour as shown in Figure 4. Although the relationship between antigen concentration and absorbance (the amount of antigen attached to the solid phase) changes depending on the procedure for adding the reagent, no matter which method is used, the relationship between antigen concentration and the amount of antigen attached to the solid phase changes. I got a relationship.

In addition, avidin (reagent 4) added by 2 in the method 2 above.
We also considered the optimal concentration of

That is, the concentration of avidin was changed to 0.5 JLg/ml, 2°0g/l, and 1.50 g/ml.

The variation of the calibration curve was examined. The results are shown in FIG.

As can be seen from Figure 5, regarding the concentration of avidin, either the absorbance increases as the concentration increases up to a certain level, or the absorbance decreases when the concentration exceeds a certain level, so there is an optimal concentration. It turned out that.

Further studies were conducted to determine the optimal concentration and applicable concentration. In method 2, the human serum albumin concentration was kept constant at 50 ng/ml, the peroxidase-labeled human serum albumin antibody concentration was kept constant at 5 ng/cod, and the biotin-labeled anti-human serum albumin concentration was 1 g/l,
2JLg/layer 1. 5kg/■1.10gg/ml, 2
At each concentration of 0 uLg/■1, the avidin concentration was changed from 0 to
As a result of varying the absorbance within a range of 2000 μg/ml and examining the variation in absorbance, the graph shown in FIG. 6 was obtained. As can be seen from Figure 6, does the most optimal avidin concentration change depending on the biotin-labeled anti-human serum albumin antibody concentration, and does the applicable range differ from the absorbance when no avidin is added? Fresh 0,5gg/+*l~2000IL
g/■l. Note that this concentration is a matter of course, and depends on the amount of biotin bound to the solid phase, the amount of biotin-labeled anti-human serum albumin, the amount of biotin added to it, the amount of avidin solution, the total amount of reaction solution, etc. affected. Therefore, the applicable avidin concentration is considered to be correlated with the above factors.

(Example 2) Figure 7 is a diagram showing the measurement principle when the present invention is applied to [competitive ETA]. Biotin B is bound to the solid phase I, and avidin A, anti-#, 2, Add biotin B labeled antigen 2A and enzyme E labeled antibody 3A, immobilize the antigen 2A antibody 3A complex on the solid phase surface of the well via avidin A, and measure the amount of enzyme (activity) remaining on the solid phase surface after washing. do.

A specific experimental example of this Example 2 will be described with reference to the results of measuring human serum albumin in the same manner as described above.

The following reagents 1 to 3 were added to a biotin-binding well prepared in the same manner as in Example 1, and reacted at room temperature for 2 hours.

Reagent 1 (antigen): Human serum albumin 50#1 (0-1
000 pg/ml) Reagent 2 (biotin-labeled antigen): biotin-labeled human serum albumin 50 pots 1 (5 tiles g/l) Reagent 3 (enzyme-labeled antibody); peroxidase-labeled anti-human serum albumin antibody 50 g I (5 g /■l) Reagent 4 (avidin): Avidin 50 River 1 (10 gg/■l) Next, reagent 4 (avidin) was added to bind the antigen-antibody complex to the solid phase surface, and after washing, the same as in Experimental Example 1 was added. The following operations were performed, the enzyme amount (activity) was measured, and a calibration curve was prepared. The results are shown in FIG. In this example experiment, reagent 2,
That is, the concentration of biotin-labeled antigen 2A is constant, and the concentration of biotin-labeled antigen 2A is constant.
However, as the concentration of antigen 2 increases, the number of bonds between reagent 3 (enzyme E-labeled antibody 3A) and biotin-labeled antigen 2A decreases. The amount of enzyme E-labeled antibody 3A that binds to will also decrease. The calibration curve in Figure 8 supports this,
I have confirmed that this method can be used.

[Comparison of reaction rates] In addition, in order to compare the reaction rates between the conventional method and the method of the present invention, a competitive EIA method was used to compare the same results! The following comparative experiment was conducted regarding lX concentration.

(Conventional method) Human serum albumin solution (
5 g/l) was added, reacted for 2 hours at room temperature, and washed to bind human serum albumin to prepare an antigen-binding plate.

Next, human serum albumin (OILgI 1-250μ
Add 50kl of g/■1) and 50g1 of peroxidase-labeled anti-human serum albumin antibody (10gg/ml), and react for 0.5 hours, 1 hour, 2 hours, 4 hours.
After washing, the same operation as in Example 1 was performed except for the time, and the enzyme amount (activity) was measured.

(The present invention) A human serum aerebumin solution (OuLg/ml to 250 g g/ml) was added to a well containing biotin at a concentration of 50.1. Add 50g of peroxidase-labeled anti-human serum albumin antibody (10ug/■1) and 50g of biotin-labeled human serum albumin (5pg/ml), and react for 0.5 hours, 1 hour, 2 hours, and 4 hours. After reaction, avidin (10 gg/ml) was added to
07zl was added and reacted for 1 hour. After washing, the same operation as in Example 1 was performed to measure the enzyme amount (activity).

Figure 9 shows the results of such a comparative experiment.1
In the case of the present invention, the absorbance after a reaction time of 0.5 hours is almost the same as in the case of a 4-hour reaction, and the reaction time is about 30 minutes or less, but according to the conventional method, the reaction does not occur even after a 4-hour reaction. It can be seen that the reaction proceeded much faster in the case of the present invention than in the case of the conventional method.

According to the conventional method, since the reaction is slow as described above, the reaction time is set to a certain period of time for measurement, but it is easily affected by temperature. On the other hand, according to the present invention, the influence of temperature is reduced and there is no need for careful time management.

[Comparison of stability] Regarding the measurement of human serum albumin in competitive EIA, we compared the stability of the conventional method explained in Figure 1O, the known method explained in Figure WSi2, and the method of the above example. , human serum albumin was measured.

Antibody 3 was bound to a solid phase in advance (for the conventional method, a reagent was prepared by the method used in the reaction rate comparison above).

Regarding the method shown in FIG. 11, avidin (20 ILg/
Intestine I) 200μ! After addition, the mixture was reacted at room temperature for 2 hours to bind avidin D, and then the plate was washed with a washing solution to prepare an adipine-bound plate.

After drying the avidin-bound plate for 2 hours at room temperature, at the time of preparation,
It was left at room temperature for 7 days after preparation and 30 days after preparation. After reacting the following reagents 1 to 3 for 4 hours in a separate test tube,
0 Ko1 was added to an avidin-binding plate, reacted for 1 hour, and after washing, the amount of enzyme was measured in the same manner as in Example 1.

Reagent 1 (antigen): Human serum albumin 100! +(0~
250 Gem+) Reagent 2 (Biotin-labeled anti-IX): Biotin-labeled human serum albumin 100 g (5 JLg/L) Reagent 3 (enzyme-labeled antibody): Veroxidase-labeled anti-human serum albumin antibody 200 g (2.5 g/meal) 1) In addition, a biotin-binding plate prepared in the same manner as in Example 1 and an antigen-binding plate prepared in the same manner as in Example 2 were left at room temperature for 7 days and 30 days after preparation. Perform the same operation, and at a reaction time of 4 hours,
Enzyme amount (activity) was measured.

Figures 12 (A) to CC) show the results of a comparative experiment of stability over time in each case of an antibody-bound plate, an avidin-bound plate, and the present example. It was observed that the absorbance of the avidin-binding plate and the antigen-binding plate decreased over time, indicating that proteins such as avidin and antigen (human serum albumin) had stability problems on the solid phase surface. Biotin is a low molecular weight compound shown by the structural formula of FIG. 13, and it was confirmed that it has excellent stability.

Although the above explanation has been made regarding the EIA method, it is clear from the above explanation that the present invention can also be applied to cases where the labeling is performed using a radioactive substance or a fluorescent substance, such as the RIA, FIA, TR-FIA, etc. .

(Effects of the Invention) The present invention is a method in which biotin is bound to a solid phase in advance, and since biotin is an organic compound, it is not susceptible to decomposition effects due to temperature, enzymes, etc. like proteins.
〈It is relatively stable and has advantages in terms of stability.

In addition, biotin is directly bound to the solid phase, and biotin-labeled antigens or antibodies are bound to this via avidin, so the solid phase prepared is a common one that binds biotin. As there is no need to prepare a solid phase for each type of antigen or antibody, the production line is simplified, production costs are reduced, and management is facilitated.

Furthermore, in the present invention, since the antigen-antibody reaction occurs in a liquid layer, the reaction rate is faster and the measurement time is shortened. In addition, it is less susceptible to the effects of temperature and time differences when measuring by setting one hour as in the conventional method, improving measurement accuracy and requiring less rough time management. It becomes easier.

[Brief explanation of drawings]

Figures 1 and 2 are diagrams of the measurement principle of the present invention, Figure 3 is a diagram of the measurement principle when the present invention is applied to sandwich EIA, and Figure 4 is the procedure for adding reagents in this embodiment regarding sandwich EIA. FIG. 5 is a diagram showing changes in the standard curve when the amount of avidin is changed in this example, and FIG. 6 is a diagram showing changes in the calibration curve when the amount of avidin is changed in this example. A diagram showing the relationship between avidin concentration and absorbance, FIG. 7 is a diagram explaining the principle when the present invention is applied to competitive EIA, and FIG. 8 is a diagram showing a calibration curve in an example of the present invention applying the EIA. Figure 9 is a comparison diagram of the measurement results of the conventional method and the method of the present invention, Figure 10 is a diagram of the measurement principle of the conventional method, Figure 11 is a diagram of the principle of a known measurement method using avidin, and Figure 12 (A ) to (C) are comparative diagrams of the stability of the conventional method, the known method, and the method according to the present invention, and FIG. 13 is the structural formula of biotin. A: Avidin, B: Biotin, E: Enzyme, ■: Solid phase, 2
．． 2A: Antigen, 3.3A: Antibody

Claims (1)

[Claims] 1. Biotin (including its analogues and derivatives) is bound in advance to the solid phase for BF separation, and at the time of measurement, biotin (including its analogues and derivatives) is bonded to the solid phase through avidin (including its analogues). , a BF separation method for immunoassay, characterized by binding an antigen or antibody labeled with biotin (including analogues and derivatives thereof). 2. In claim 1, the avidin concentration is 0.5 μg.
1. A BF separation method for immunoassay, characterized in that the concentration is 2000 μg/ml to 2000 μg/ml.