JPH07159406A - Cleaning liquid and cleaning method - Google Patents

Abstract

(57) [Summary] [Objective] When performing an immunoassay in a reaction container, it is possible to reduce the background value caused by nonspecific adsorption of the labeling substance to the reaction container and the carrier on which the immune component is immobilized. A cleaning liquid and a cleaning method are provided. A cleaning solution containing a sugar-based nonionic surfactant and a substance having a chaotropic ion, and a cleaning method characterized by increasing the amount of the cleaning solution to be used step by step achieve the above object.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a washing solution and a washing method used in an immunoassay utilizing an antigen-antibody reaction. More specifically, the present invention relates to an immunological reaction using an inner wall of a reaction vessel or an insoluble carrier as a solid phase. The present invention relates to a washing solution for washing a reaction system containing a solid phase and a method for washing the solid phase after the completion of an antigen-antibody reaction when measuring a highly active substance.

[0002]

2. Description of the Related Art There are many known methods for analyzing biological components. However, when analyzing a specific trace amount of a mixed component, a highly specific and highly sensitive method is required. Desired. Recently, from this viewpoint, many analytical methods utilizing biological affinity have been carried out. For example, 19
The radioimmunoassay (RIA) reported by Berson and Yalow in 1959 has a higher detection sensitivity and specificity than chemical and bioassay methods and immunological methods used until then. The law was in the limelight and opened the door to a new field of immunoassay (R.S. Yalo
w et al., Nature, 184, 1648 (1959)).

However, since the RIA uses a radioactive substance (RI) as a labeling substance and requires a special facility and a measuring device, various non-radioactive immunoassay methods have been studied in recent years. Enzyme Immunoassay (EIA), which was independently reported by Engvall et al. And Weemen et al.
A method of labeling an enzyme with an antigen or an antibody and quantifying the concentration of the antigen or the antibody in the sample from the enzyme activity value. The enzyme-antibody method that has been used in histochemistry up to that time is applied to the reaction in a liquid phase system based on the principle of RIA. It was used.

EIA is similar to RIA in principle, but various devises have been made in the measurement because an enzyme, which is a high molecular protein, is used as a labeling substance. So far, the measurement target, detection type, reaction phase, etc. are different. A number of methods have been proposed. For example, in the reaction between an enzyme-labeled antigen (antibody) and an antibody (antigen), there are a competitive method and a non-competitive method depending on whether or not the unlabeled antigen or antibody competes. In addition, there are a separation method (heterogeneous method) and a non-separation method (homogeneous method) depending on the measurement method of the antigen / antibody reaction product. The former separates the antigen / antibody reaction product and the unreacted product. Then, measure the labeled enzyme. Note that there are two methods for separating the antigen-antibody reaction in the liquid phase (liquid phase method) and a method between the liquid phase and the solid phase (solid phase method), but the liquid phase method requires centrifugation. Therefore, the solid phase method is generally used.

For example, in the sandwich method, the inner wall of the reaction vessel and the solid phase such as an insoluble carrier are coated with an antibody or the like in advance, and the antigen or the like in the sample is reacted therewith, followed by detection using an enzyme-labeled antibody or the like. Usually, beads, microplates, tubes, etc. are used as the carrier, but recently fine particles (magnetic particles etc.) may be used for the purpose of shortening the reaction time.

[0006]

In the heterogeneous immunoassay method, after the antigen-antibody reaction, the bound portion of the antigen-antibody conjugate and the unreacted free portion are separated, and the bound portion or the released component is labeled. It measures the amount of trace substances and has a higher sensitivity compared to the homogeneous immunoassay method, and can detect ultratrace amounts (1 atto mol
Measurement). On the other hand, samples such as serum contain a large amount of non-specific immunoglobulins, which are non-specifically adsorbed on the solid phase, which significantly increases the background and interferes with high-sensitivity measurement. . As a measure to reduce this non-specific adsorption, we devised an IgG-labeled antibody (adaptation of hinge method), an immunocomplex transfer assay (Kohno, T., Ishik).
awa, E., Biochem.Biophys.Res.Commun., 147,644,1987
), The material of the equipment of the solid carrier has been improved.

[0007]

[Means for Solving the Problems] The inventors of the present invention inject a washing solution into a reaction container in which an immune reaction has been carried out for the purpose of preventing an increase in background resulting from non-specific adsorption on the solid phase. As a result of diligently studying the cleaning operation of sucking and removing after that, non-specific adsorption can be reduced by devising the components of the cleaning liquid or the amount of the cleaning liquid used, and as a result, the background can be reduced. Found and completed the present invention. That is, the present invention forms a solid phase component containing the substance to be measured by contacting the inner wall of the reaction vessel or the insoluble carrier on which the immune reaction component that specifically binds to the substance to be measured in the sample is immobilized, with the sample, Immune reaction component labeled with a detectable substance that is washed by a first washing operation consisting of injecting a washing liquid after separating the solid-phase component from the liquid-phase component, aspirating / removing, and binding specifically to the substance to be measured Alternatively, it is reacted with a substance that is immunologically equivalent to the substance to be measured labeled with a detectable substance to form a solid phase component containing the detectable substance, and the solid phase component is separated from the liquid phase component to be a washing solution. The second method in the method for measuring a substance to be measured in the sample by washing by a second washing operation, which comprises injecting, aspirating and removing, and detecting a detectable substance in a solid phase or a liquid phase component. First and / or second wash A cleaning liquid to be used in the operation, a cleaning fluid which is characterized by containing a substance having a sugar-based nonionic surfactants and chaotropic ions.

The present invention also provides a detectable substance that specifically binds to an inner wall of a reaction container or an insoluble carrier on which an immune reaction component that specifically binds to a substance to be measured in a sample is immobilized, to the sample and the substance to be measured. And a sample and a substance to be measured labeled with a detectable substance are immunologically equivalent to form a solid phase component containing the detectable substance. After the component is separated from the liquid phase component, a washing solution is injected, and the sample is washed by a washing operation consisting of suction and removal, and the substance to be measured in the sample is detected by detecting a detectable substance in the solid or liquid phase component. A cleaning liquid used in the cleaning operation in the measuring method,
A cleaning solution containing a sugar-based nonionic surfactant and a substance having chaotropic ions.

In the present invention, the inner wall of the reaction vessel or the insoluble carrier on which the immune reaction component that specifically binds to the substance to be measured in the sample is immobilized is brought into contact with the sample to form a solid phase component containing the substance to be measured. An immunization labeled with a detectable substance which is washed by a first washing operation consisting of injecting, aspirating and removing a washing solution after separating the solid phase component from the liquid phase component and specifically binding to a substance to be measured. A solid phase component containing the detectable substance is formed by reacting with a substance that is immunologically equivalent to the measurement target substance labeled with the reaction component or the detectable substance, and the solid phase component is separated from the liquid phase component. A method of measuring a substance to be measured in the sample by washing by a second washing operation consisting of injecting, aspirating and removing a post-washing liquid, and detecting a detectable substance in a solid phase or a liquid phase component Yes, the first and Alternatively, the second cleaning operation is performed by injecting, aspirating / removing a cleaning liquid in an amount substantially equal to that of the liquid phase component separated immediately before, and injecting, aspirating / removing a larger amount of the cleaning liquid than the cleaning liquid used in the operation. It is a method carried out by.

Further, the present invention provides a detectable substance that specifically binds to the sample and the substance to be measured, by using the inner wall of the reaction vessel or the insoluble carrier on which the immunoreactive component that specifically binds to the substance to be measured in the sample is immobilized. The immunoreactive component labeled with or the sample and the substance to be measured labeled with a detectable substance are reacted with a substance which is immunologically equivalent to form a solid phase component containing the detectable substance, and the solid phase After the component is separated from the liquid phase component, a washing solution is injected, and the sample is washed by a washing operation consisting of suction and removal, and the substance to be measured in the sample is detected by detecting a detectable substance in the solid or liquid phase component. A method of measuring, in which the washing operation is performed by injecting approximately the same amount of the washing liquid as the liquid phase component separated immediately before that, aspirating / removing operation, and injecting and aspirating a larger amount of the washing liquid than the washing liquid used in the operation. By removing operation Is a method to be applied. The present invention will be described in detail below.

The substance to be measured means an immunologically active substance such as an antigen or an antibody, and the assay itself may be a sandwich method or a competitive method. Further, it may be a one-step method in which the solid phase and the liquid phase are separated once, or a two-step method in which the solid phase and the liquid phase are separated twice. Further, in the present invention, a solid phase in which an antibody or the like is bound to the surface of an insoluble carrier prepared separately from the reaction container may be used, or the insoluble carrier is not used and the antibody or the like is bound to the inner wall of the reaction container. You may let me. The cleaning liquid or the cleaning method of the present invention is a cleaning liquid or a cleaning method for simultaneously cleaning the inner wall of the reaction container and the solid phase, and the effect can be achieved regardless of the case of the solid phase. Detectable substances used as labels include fluorescent substances, luminescent substances, colored substances, light-absorbing substances, radioisotopes, etc., as well as substances such as enzymes that cannot be detected (difficult) themselves. Can be used without any particular limitation, such as one that can cause a detectable chemical reaction.

To describe the two-step sandwich method as an example, first, the antigen in the sample is bound to the immobilized antibody, and the unbound liquid phase component (sample or the like) is sucked and removed. Next, an enzyme-labeled antibody is added to bind to the antigen on the solid phase, the antigen to be measured is sandwiched between the immobilized antibody and the labeled antibody, and the unbound labeled antibody is aspirated and removed. Then, a washing operation is performed by injecting an appropriate washing solution and aspirating / removing so that there is no residual labeled antibody in the reaction vessel, and finally, in the liquid phase bound to the solid phase or aspirated / removed with the washing solution. The activity of the enzyme is measured to determine the amount of antigen.

The washing solution of the present invention is used in a washing operation after separation of liquid phase components in the above-mentioned assay, and contains a sugar-based nonionic surfactant and a substance having a chaotropic ion, It has the effect of aspirating and removing the labeled antibody or the like non-specifically adsorbed to the solid phase component without affecting the immune binding of the solid phase component. The cleaning solution of the present invention may contain various buffer agents, components such as sodium chloride, which are usually used as cleaning solution components. The cleaning liquid of the present invention can be adjusted in pH by using an alkaline or acidic chemical,
It is preferably 2 to 10, particularly preferably 4 to 8.

Examples of the sugar-based nonionic surfactant include alkyl saccharide-based surfactants represented by R ₁ -O-Gn (R ₁ is a linear or branched alkyl group having 6 to 18 carbon atoms). , Alkenyl or alkylphenyl group, and n represents a number of 1 to 10), among which,
Twelve linear or branched alkyl groups are preferred in terms of cleaning properties. Further, the saccharide moiety (G) which is a hydrophilic group has a reducing sugar having 5 to 6 carbon atoms as a basic unit, and glucose, galactose and fructose are preferable as the reducing sugar. The average degree of polymerization (n) of the saccharide is 1 to 10, preferably 1 to 4 and particularly preferably n = 1. Considering the influences of both the average degree of polymerization and the R ₁ group on the properties of the compound, when R ₁ has 8 carbon atoms, n = 1 is preferable. Examples of preferred sugar-based nonionic surfactants that can be used in the present invention include octyl β-glucoside, hexyl β-glucoside, and n-dodecyl-β-D-maltoside. The concentration in the cleaning liquid is 0.001 to 1.0% (weight / volume), preferably 0.01 to 0.1% (weight / volume).

As the substance having a chaotropic ion, a substance having a weak denaturing action to the extent that it does not inactivate proteins such as antigens and antibodies is preferable, but it has the property of inactivating proteins at a certain concentration or more. Also, it can be used within a concentration range below the concentration at which antigens, antibodies, etc. involved in the actual reaction are not inactivated. Examples thereof include calcium chloride, thiocyanate (potassium salt, sodium salt), lithium bromide, guanidine hydrochloric acid, guanidine thiocyanic acid and the like, and among these, thiocyanate, lithium chloride and calcium chloride are particularly preferable. These substances having chaotropic ions can be used alone or in combination of two or more kinds.
0.0001-0.1% (weight / volume) as concentration in cleaning solution
Is preferable, but particularly preferably 0.001 to 0.01% (weight /
Capacity).

Conventionally, washing is carried out by injecting a fixed amount of washing liquid into a reaction vessel to dilute the remaining liquid phase component, and sucking and removing it. Generally, the reaction volume used in immunoassay is about 200-500 μl,
After the immune reaction is completed and the liquid phase is separated, a large amount of washing liquid is injected as compared with the amount. This is because the purpose is to enhance the cleaning effect by diluting the remaining components with the cleaning liquid. The washing effect at this time is uniquely determined by the dilution rate, but when a large amount of washing liquid is used for the first injection, aspiration and removal of the washing liquid, the remaining liquid phase, especially the high-concentration labeled substance, is So that it contacts the top wall of the container.
Therefore, the range of contamination by the component is expanded.

In the method of the present invention, after the liquid phase component is separated from the reaction liquid, first, a washing liquid of approximately the same amount as the separated liquid phase component is injected, and suction / removal operations are performed, and then the liquid liquid component is used in the operation. The feature is that a larger amount of cleaning liquid than the cleaning liquid is injected, and suction / removal operations are performed. Do the washing operation twice,
In the so-called two-step method, a substance that can be detected in the second washing operation is used. Therefore, in such a two-step sandwich or competitive method, it is particularly preferable to carry out the second washing operation according to the present invention.

In the method of the present invention, first, an operation of injecting and aspirating / removing a cleaning liquid in an amount substantially equal to that of the liquid phase component separated immediately before (hereinafter referred to as the first operation) is carried out, and then the cleaning liquid used in the above operation is removed. Injection of a large amount of cleaning liquid, suction / removal operation (hereinafter referred to as the second
Operation). The amount of the cleaning liquid used in the second operation may be, for example, slightly larger than the amount of the cleaning liquid in the first operation, but it is particularly preferable to set the maximum amount that can be injected into the reaction container in order to increase the dilution rate. Of course, as the second operation, the maximum amount of the cleaning liquid that can be injected into the reaction vessel may be injected, or the amount of the cleaning liquid to be gradually injected may be increased, or the first operation may be repeated a plurality of times before the second operation. You may go. In particular, it is preferable to perform the first operation using the same amount of the cleaning liquid as the separated liquid phase component, and then to perform the second operation a plurality of times in which the amount of the cleaning liquid is increased step by step.

By the way, after the final washing operation is completed, the solid phase component is further brought into contact with an immune reaction component such as a labeled antibody or a reaction solution for measuring a detectable substance. Therefore, in order to prevent the cleaning liquid from remaining on the inner wall of the reaction vessel at the end of the cleaning operation, first of all, injecting, aspirating, and aspirating the cleaning liquid in an amount substantially the same as the liquid phase component separated immediately before that.
Perform the removal operation (the first operation), then inject a larger amount of the cleaning solution than the cleaning solution used in the first operation, perform the suction / removal operation (the second operation), and further perform the maximum operation used up to that point. The amount of cleaning liquid that gradually decreases from the amount of cleaning liquid
It is advisable to perform a plurality of washing operations for suction / removal (hereinafter referred to as the third operation).

Since the cleaning liquid adhering to the inner wall of the reaction vessel by the cleaning operation up to the second operation can exist only at or below the point where the liquid level is located when the maximum cleaning solution is used even at the highest position, from this point It is possible to prevent the cleaning liquid from remaining on the inner wall by repeating the operation of injecting the cleaning liquid so that the liquid surface reaches a slightly lower point and absorbing the residual liquid. The amount of the cleaning liquid used at this time can be exemplified to decrease according to a bell-shaped curve having the maximum amount of the cleaning liquid used in the operations up to that point as a peak, but it may be decreased stepwise.

In the above-described method of the present invention, the washing liquid containing the above-described sugar-based nonionic surfactant and the substance having chaotropic ions is used as the washing liquid, whereby nonspecific adsorption to the solid phase component is achieved. Can also be prevented.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Dried 3.86 g of a spherical polystyrene (1.2 mm in diameter) containing a weighed amount of ferrite was dispersed in 7 ml of an anti-human TSH monoclonal antibody solution and stirred under room temperature conditions. Leave for 20 hours, then 20 mM Tri-H containing 0.2 M NaCl
It was washed with Cl buffer (pH 6.8) and masked with 1% BSA (bovine serum albumin) to prepare an antibody-immobilized carrier (solid phase).

Example 2 Using various cleaning solutions, their cleaning effects were tested. 10 of the antibody-immobilized carriers prepared in Example 1 were placed in a polypropylene reaction container (upper inner diameter 10 mm, bottom inner diameter 8 mm), and 100 μl of the substance to be measured (negative human serum containing no TSH or a fixed amount) Amount of human serum containing TSH) and
50 μl of alkaline phosphatase-conjugated anti-human TSH monoclonal antibody (recognizing a different epitope than the monoclonal antibody in Example 1) was added.
The mixture was stirred for 20 minutes at 37 ° C. to generate an immune reaction.

After completion of the reaction, the components not bound to the solid phase (liquid phase components) were removed by suction, 0.75 ml of the cleaning liquids of various compositions were injected, and the cleaning operation of suction removal was carried out once. As a control, a washing operation with 20 mM Tris-HCl (pH 7.0) was also performed.

After completion of the washing operation, an alkaline phosphor
Solution containing adamantyl methoxyphosphoryl phenyldioxetane, a substrate for ze (Lumiphos-530, Wako Pure Chemical Industries)
50 μl was added, and luminescence for 3 minutes from that point was detected using a commercially available luminescence detector (BLR-301, manufactured by Aloka).

The results are shown in Table 1. In the table, NaSCN is sodium thiocyanate, CaCl2 is calcium chloride, and LiBr.
Is lithium bromide, NaCl is sodium chloride, Tween 20
Is Tween 20, Triton X-100 is Triton X-100, OB
G indicates octyl β-glucoside, HBG indicates hexyl β-glucoside, and the concentration of each composition is Tween 20, TritonX-
100, OBG and HBG are 0.01%, NaSCN, CaCl2,
LiBr and NaCl are 0.01M.

In the table, the luminescence value (Kcpm) in the negative serum containing no TSH is a so-called background value, and it is desired that the value is small. On the other hand, since the sample serum is the same in all cases, it is desired that the sample serum has a large luminescence value. According to the table, when a washing solution containing a sugar-based nonionic surfactant such as OBG or HBG and a chaotropic ion such as NaSCN or CaCl2LiBr is used, the background value decreases but the luminescence value in the sample serum remains large. It turns out that

[0029]

[Table 1]

Example 3 Containing 0.08% NaSCN, 0.01% OBG and 0.1M NaCl,
Using 20 mM Tris-HCl (pH 7.0) solution as a washing solution, TSH
A sandwich immunoassay was performed using negative human serum as a sample. As a control, 0.015% Tween-20, 0.1 M NaCl
The same measurement was performed using a 20 mM Tris-HCl (pH 7.0) solution containing

Polypropylene reaction vessel (upper inner diameter 10
mm, bottom inner diameter 8 mm), 12 of the antibody-immobilized carriers prepared in Example 1 were added, and 100 μl of the substance to be measured (negative human serum containing no TSH or human serum containing a certain amount of TSH) And 50 µl of alkaline phosphatase-conjugated anti-human TSH monoclonal antibody (recognizing a different epitope than the monoclonal antibody in Example 1)
Was added and the mixture was stirred for 20 minutes at 37 ° C. to induce an immune reaction.

After the completion of the reaction, the components not bound to the solid phase (liquid phase components) were removed by suction, 1 ml of the washing solution was injected, and the washing operation of removing by suction was carried out four times.

After the washing operation is completed, an alkaline phosphator is used.
Solution containing adamantyl methoxyphosphoryl phenyldioxetane, a substrate for ze (Lumiphos-530, Wako Pure Chemical Industries)
50 μl was added, and luminescence for 3 minutes from that point was detected using a commercially available luminescence detector (BLR-301, manufactured by Aloka).

The results are shown in Table 2. In the table, the luminescence value (Kcpm) in the negative serum containing no TSH is a so-called background value, and it is desired that the value is small.
On the other hand, it is desired that the sample serum has a large luminescence value. According to the table, an improvement in the washing effect was observed even when the control washing solution was used after four washing operations, and the luminescence value of the sample serum was almost the same as that of the washing solution of the present invention, but the luminescence value of the negative serum ( Since the background value) is large, it can be seen that the S / N ratio is inferior to the cleaning solution of the present invention. Further, it can be seen that the variation value (CV%) calculated from 5 trials is large as compared with the cleaning liquid of the present invention.

[0035]

[Table 2]

Example 4 Containing 0.08% NaSCN, 0.01% OBG and 0.1M NaCl,
Using 20 mM Tris-HCl (pH 7.0) solution as a washing solution, TSH
A sandwich immunoassay was performed using negative human serum as a sample.

Polypropylene reaction vessel (upper inner diameter 10
mm, bottom inner diameter 8 mm), 12 of the antibody-immobilized carriers prepared in Example 1 were added, and 100 μl of the substance to be measured (negative human serum containing no TSH or human serum containing a certain amount of TSH) And 50 µl of alkaline phosphatase-conjugated anti-human TSH monoclonal antibody (recognizing a different epitope than the monoclonal antibody in Example 1)
Was added and the mixture was stirred for 20 minutes at 37 ° C. to induce an immune reaction.

After completion of the reaction, the components not bound to the solid phase (liquid phase components) were removed by suction, and the washing operation was carried out by the following two washing methods (1) or (2).

Washing method (1) Inject 0.75 ml of washing liquid,
Washing procedure in which suction removal is simply repeated 4 times Washing method (2) First operation using 0.25 ml of washing solution, second operation using 0.75 ml of washing solution, third operation using 0.75 ml of washing solution After the washing operation, 50 µl of a solution containing adamantyl methoxyphosphorylphenyl dioxetane (Lumiphos-530, manufactured by Wako Pure Chemical Industries, Ltd.) which is a substrate of alkaline phosphatase is used. In addition, the commercially available luminescence detector (BL
R-301, manufactured by Aloka).

The results are shown in Table 3. According to the table, the composition of the washing solution is the same for both the luminescence value of the negative serum (background value) and the luminescence value of the sample serum, and the amount of the washing solution used is smaller than that of the washing method (1) by about 1 ml. In other words, the washing method (2) can obtain a better washing effect, that is, a smaller luminescence value in the negative serum and a larger luminescence value in the sample serum, and further, an effect that both values have small fluctuation values can be achieved. I understand.

[0041]

[Table 3]

[0042]

EFFECTS OF THE INVENTION According to the cleaning solution of the present invention, a reaction container for carrying out an immunoreaction and / or a carrier on which an antibody or the like used in a heterogeneous method is immobilized is immobilized on a carrier which does not contain an immunological component or the like bound to a labeling substance. Specific adsorption can be reduced. As a result, the background value in the immunoassay can be reduced, and as a result, the immunoassay with higher sensitivity than in the past can be realized.

According to the cleaning method of the present invention, the effect of reducing the background value can be achieved with a smaller amount of cleaning liquid.

Claims (9)

[Claims] 1. A solid phase component containing a substance to be measured is formed by contacting the inner wall of a reaction vessel or an insoluble carrier, on which an immune reaction component that specifically binds to the substance to be measured in a sample is immobilized, with a sample to form a solid phase component. After the phase component is separated from the liquid phase component, the washing solution is injected by a first washing operation consisting of injecting, aspirating and removing, and an immune reaction component labeled with a detectable substance that specifically binds to the substance to be measured or A solid phase component containing the detectable substance is formed by reacting with a substance that is immunologically equivalent to the substance to be measured labeled with the detectable substance, and the solid phase component is separated from the liquid phase component to obtain a washing solution. The first method in the method for measuring a substance to be measured in the sample by washing by a second washing operation including injection, suction and removal, and detecting a detectable substance in a solid phase or a liquid phase component And / or a second washing operation A cleaning liquid used for the production, which contains a sugar-based nonionic surfactant and a substance having a chaotropic ion. 2. An inner wall of a reaction vessel or an insoluble carrier on which an immune reaction component that specifically binds to a substance to be measured in a sample is immobilized is labeled with a detectable substance that specifically binds to the sample and the substance to be measured. Immunoreactive component or sample and a substance that is immunologically equivalent to the substance to be measured labeled with a detectable substance to form a solid phase component containing the detectable substance, and the solid phase component is a liquid. A method of measuring a substance to be measured in the sample by washing by a washing operation consisting of injecting, aspirating / removing a washing liquid after separating the phase component from the solid component or the liquid phase component to detect a detectable substance A cleaning liquid used in the above-mentioned cleaning operation in 1., which contains a sugar-based nonionic surfactant and a substance having a chaotropic ion. 3. The cleaning liquid according to claim 1, wherein the substance having chaotropic ions is one or more substances selected from the group consisting of thiocyanate, calcium chloride and lithium chloride. 4. A solid phase component containing a substance to be measured is formed by contacting an inner wall of a reaction vessel or an insoluble carrier, on which an immune reaction component that specifically binds to the substance to be measured in the sample is immobilized, with the sample to form a solid phase component. After the phase component is separated from the liquid phase component, the washing solution is injected by a first washing operation consisting of injecting, aspirating and removing, and an immune reaction component labeled with a detectable substance that specifically binds to the substance to be measured or A solid phase component containing the detectable substance is formed by reacting with a substance that is immunologically equivalent to the substance to be measured labeled with the detectable substance, and the solid phase component is separated from the liquid phase component to obtain a washing solution. A method for measuring a substance to be measured in the sample by washing by a second washing operation comprising injection, suction and removal, and detecting a detectable substance in a solid phase or a liquid phase component, The first
And / or the second washing operation is an operation of injecting, aspirating / removing the washing liquid of substantially the same amount as the liquid phase component separated immediately before that,
The method as described above, which is carried out by injecting and aspirating / removing a larger amount of the cleaning liquid than the cleaning liquid used in the operation. 5. An inner wall of a reaction vessel or an insoluble carrier on which an immune reaction component that specifically binds to a substance to be measured in a sample is immobilized is labeled with a detectable substance that specifically binds to the sample and the substance to be measured. Immunoreactive component or sample and a substance to be measured that is labeled with a detectable substance and is immunologically equivalent to form a solid phase component containing the detectable substance, and the solid phase component is a liquid. A method of measuring a substance to be measured in the sample by washing by a washing operation consisting of injecting, aspirating / removing a washing liquid after separating the phase component from the solid component or the liquid phase component to detect a detectable substance The cleaning operation is performed by injecting and aspirating / removing a cleaning liquid in an amount substantially equal to that of the liquid phase component separated immediately before, and injecting and aspirating / removing a larger amount of cleaning liquid than the cleaning liquid used in the operation. The above is carried out Method. 6. Immediately after the injection, suction / removal operation of a cleaning liquid of approximately the same amount as the liquid phase component separated immediately before that, and the injection / aspiration / removal operation of a cleaning liquid in a larger amount than the cleaning liquid used in the operation. 6. The method according to claim 4 or 5, wherein a plurality of cleaning operations are performed, in which an amount of the cleaning liquid that gradually decreases from the maximum amount of the cleaning liquid used in the above operation is injected, and suction / removal is performed. 7. A plurality of washing operations of injecting, aspirating / removing a gradually increasing amount of the washing liquid after injecting, aspirating / removing the same amount of the washing liquid as the liquid phase component separated immediately before that. The method according to claim 4, wherein the method is performed. 8. The method according to claim 4, wherein a cleaning liquid containing a sugar-based nonionic surfactant and a substance having chaotropic ions is used. 9. The method according to claim 8, wherein the substance having chaotropic ions is one or more substances selected from the group consisting of thiocyanate, calcium chloride and lithium chloride.