JP2000249705A - Alkaline wash for automatic immunoassay, and immunoassay method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a wash which improves in washing efficiency and enables higher sensitive measurement in immunoassay using an automatic immunoassay device and to provide an immunoassay method using it. SOLUTION: This wash for automatic immunoassay comprises an alkaline aqueous solution containing two or more kinds of surfactant selected from (1) a polyoxyalkylene alkyl ether-based nonionic surfactant (2) a polyoxyalkylene allyl ether-based nonionic surfactant (3) an alkyl diallyl ether sulfonate-based anionic surfactant. This immunoassay method employs this alkaline wash in a washing process of an automatic immunoassay method where an antigen or antibody in a specimen is measured through the use of an antigen-antibody reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an alkaline washing solution for immunoassay using an automatic immunoassay apparatus and an immunoassay method using the same.

[0002]

2. Description of the Related Art An immunoassay method using a reaction in which fine particles such as polystyrene latex aggregate with an immune reaction is widely used as a quick, simple, and accurate measurement method. An automatic immunoassay device that automates the measurement procedure and improves the processing efficiency has also been developed. In an automatic immunoassay apparatus, a measurement container, a dispensing nozzle for injecting a specimen or a reagent into the measurement container, a stirring probe for stirring the reaction solution in the measurement container, and the like are usually used repeatedly. For this reason, if these washings are not performed sufficiently during the measurement procedure, the reaction solution components (reagents, specimens, etc.) are brought into the next measurement, which affects the measured values.

[0003]

In order to avoid the influence of the carry-in, conventionally, an alkaline cleaning solution containing a surfactant has been used. However, depending on the types of specimens and reagents, and combinations thereof, the conventional cleaning solution was sometimes insufficient.

[0004]

The inventors of the present invention have conducted intensive studies on the components of this cleaning solution, and have found that by using two or more specific surfactants in combination,
The present inventors have found that the cleaning effect is significantly improved, and have reached the present invention. That is, the gist of the present invention is as follows: (a) to (c) (a) a polyoxyalkylene alkyl ether-based nonionic surfactant (b) a polyoxyalkylene aryl ether-based nonionic surfactant (c) alkyl diallyl ether Measurement of an antigen or antibody in a sample by a washing solution for automatic immunoassay characterized by comprising an alkaline aqueous solution containing two or more surfactants selected from sulfonate-based anionic surfactants, and an antigen-antibody reaction The present invention also provides an immunoassay method comprising using the alkaline washing solution in the washing step of the automatic immunoassay method.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The cleaning liquid of the present invention is characterized by containing two or more specific surfactants. The polyoxyalkylene alkyl ether-based nonionic surfactant (a) used in the present invention has an HLB value of 7 to 20,
Formula _{R-O- (CH 2 CH 2} O) n -H ( wherein, R generally having 10 to 22, preferably 12 to
22 alkyl groups, n is usually 3 to 100, preferably 3
Represents a number from 50 to 50. However, nonionic surfactants represented by R and n are average values). As such a surfactant, for example, trade name NIKKOL BO-
10TX (R = C ₁₀ H ₂₁ , n = 10), Emulgen 12
0 (R = C ₁₂ H ₂₅ , n = 20), Brij 35 (R = C
₁₂ H ₂₅ , n = 23), Brij 58, Emulgen 220
(R = C ₁₆ H ₃₃ , n = 20), NIKKOL BC-2
3TX (R = C ₁₆ H ₃₃ , n = 23), Brij78, Emulgen 320P (R = C ₁₈ H ₃₇ , n = 20), Bri
j700 (R = C ₁₈ H ₃₇ , n = 100), NIKKOL
Can be used those commercially available as _{BB-30 (R = C 22} H 45, n = 30) or the like. Among these, R is an alkyl group having 12 to 18 carbon atoms, and n is 2
Those having 0 to 25 are particularly preferred.

The polyoxyalkylene aryl ether nonionic surfactant (b) used in the present invention includes:
HLB value is 7-20, the general formula _{R-Ph-O- (CH 2} CH 2 O) n -H ( wherein, R is an alkyl group of 8 to 9 carbon atoms, Ph is a phenyl group, n represents 3 And a nonionic surfactant represented by the formula (1), wherein R and n are average values. Such surfactants include, for example, trade name Trit
onX-100 (R = C ₈ H ₁₇ , n = 9-10), Emulgen 810 (R = C ₈ H ₁₇ , n = 10), Trito
_{nX-165 (R = C 8} H 17, n = 16), Trito
_{nX-405 (R = C 8} H 17, n = 40), NP-20
(R = C ₉ H ₁₉ , n = 20), Emulgen 903 (R =
C ₉ H ₁₉ , n = 3), Emulgen 905 (R = C
₉ H ₁₉ , n = 5), Emulgen 906 (R = C ₉ H ₁₉ ,
n = 6), Emulgen 909 (R = C ₉ H ₁₉ , n =
9), Emulgen 910 (R = C ₉ H ₁₉ , n = 10),
Emulgen 913 (R = C ₉ H ₁₉ , n = 13), Emulgen 920 (R = C ₉ H ₁₉ , n = 20), Emulgen 9
30 (R = C ₉ H ₁₉ , n = 30), Emulgen 935
(R = C ₉ H ₁₉ , n = 35), Emulgen 950 (R =
C ₉ H ₁₉ , n = 50), Emulgen 985 (R = C ₉ H
₁₉ , n = 85), Emarex OP (R = C ₈ H ₁₇ ),
A commercially available product such as Emarex NP (R = C ₉ H ₁₉ ) can be used. Among these, R
Is an alkyl group having 8 to 9 carbon atoms, and a surfactant in which n is 9 to 20 is particularly preferable. Specifically, the product name T
ritonX-100, Emulgen 810, Trito
Preferred are nX-165, NP-20, Emulgen 910, Emulgen 913, Emulgen 920 and the like.

The alkyl diallyl ether sulfonate-based anionic surfactant (c) used in the present invention includes:
It is preferable that the alkyl group has 8 to 18 carbon atoms.
As such an anionic surfactant, for example, those marketed under the trade names Perex SS-L, Perex SS-H, etc. can be used.

The present invention is characterized in that two or more of the above-mentioned surfactants are used in combination, and the method of using the same is usually a method of combining different kinds of the above (a) to (c), or Any method of combining two or more of the same types may be used. When two or more surfactants of the same type (a) or (b) are combined, it is usually necessary to use a combination of surfactants having different alkyl ether structures and aryl ether structures. Specific examples include the combinations shown in Table 1 below.

[0009]

[Table 1]

Particularly preferred combinations include, for example, (a) two kinds, (a) one kind and (b) one kind, and (b)
There are two types. Specifically, the product name Brij7
8 and Triton X-100, NP-20 and Trito
nX-100, Brij35 and TritonX-10
0, Brij58 and TritonX-100, NIKK
OL BO-10TX and Triton X-100, NI
A combination of KKOL BC-23TX and Triton X-100 is particularly preferred.

The mixing ratio of the two surfactants is usually 1: 9 to 9: 1 by weight, preferably 1: 3 to 3:
It is one. The concentration of the surfactant in the washing solution is usually 0.02 to 2% by weight, preferably 0.06 to 0.6%.
% By weight, and the concentration of each surfactant is 0.1%.
01% by weight or more, preferably 0.03% by weight or more, more preferably 0.05% by weight or more, and 1% by weight
Or less, preferably 0.3% by weight or less. Further, three or more surfactants may be used in combination. When three surfactants are used, the mixing ratio is usually from 18: 1 to 2: 9, preferably from 6: 1 to 2: 3, by weight, based on the total of the two surfactants. As a combination of three kinds of surfactants, (a) two kinds and (b) one kind, or (a) one kind
Species and (b) two are preferred.

The cleaning solution of the present invention is an aqueous alkaline solution containing the above-mentioned surfactant. Examples of the alkali component include metal hydroxides, preferably alkali metal hydroxides such as sodium hydroxide and potassium hydroxide. The amount of the alkali component in the cleaning solution is usually 0.01 to
It is 5 normal, and preferably 0.05 to 1 normal.

The immunoassay method of the present invention is based on an automatic immunoassay apparatus, and includes, for example, the following methods. (1) A method in which a specimen, an immunoreagent containing an antibody or an antigen, and a liquid medium (reaction buffer) are dispensed into a measurement container, stirred, and agglutination of particles due to the antigen-antibody reaction is measured by absorbance or scattered light. (2) The method according to (1), wherein the immunoreagent is an immunoreagent in which an antibody or an antigen is immobilized on insoluble carrier particles. (3) Sample, first immunoreagent having antibody or antigen immobilized on magnetic particles, second immunoreagent containing antibody or labeled antigen labeled with a labeling agent, and liquid medium (reaction buffer) After dispensing into a measurement container, stirring is performed, and then a magnetic field is applied to the reaction mixture to separate and wash unreacted magnetic particles and aggregated particles containing the magnetic particles. How to measure the concentration. (4) Sample, first immunoreagent in which antibody or antigen is immobilized on magnetic particles, second immunoreagent containing antibody or labeled antigen labeled with a labeling agent, and liquid medium (reaction buffer) Is dispensed into a measuring container, stirred, and then a magnetic field is applied to the reaction mixture to separate unreacted magnetic particles and aggregated particles containing magnetic particles, and measure the concentration of the labeling agent not involved in the antigen-antibody reaction. how to. (5) The second immunoreagent is one in which a labeling agent is directly bound to an antibody or antigen, or the one in which an antibody or antigen is immobilized on insoluble particles containing a labeling agent (3)
Or the method of (4).

In the above methods (3) to (5), a separation and washing step may be inserted between the first reaction with the first immunoreagent and the second reaction with the second immunoreagent. You do not have to enter. The first and second immunological reagents may be dispensed simultaneously. In addition, when the separation and washing step is not performed between the first reaction and the second reaction, the second reagent may be used for the first reaction, and the first reagent may be used for the second reaction. . As a method for detecting the labeling agent, fluorescence, chemiluminescence, electrochemiluminescence, or the like can be used.

The procedure of the automatic immunoassay is as follows, taking the method (3) as an example. (S1) A liquid medium and a sample are injected into a measurement container using a dispensing nozzle. (S2) Inject the magnetic particles on which the antibody or antigen is immobilized as the first immunoreagent into the measurement container. (S3) The inside of the measurement container is stirred. (S4) A magnetic field is applied to the measurement container after the reaction to separate unreacted magnetic particles and aggregated particles containing magnetic particles from a liquid medium containing other components not involved in the antigen-antibody reaction. (S5) Drain the liquid medium. (S6) Remove the measurement container from the magnetic field. (S7) A second immunoreagent containing a labeled antibody or a labeled antigen is injected into a measurement container together with a liquid medium. (S8) Stir the inside of the measurement container and wait for the reaction to proceed sufficiently. (S9) A magnetic field is applied again to separate the labeled antibody or labeled antigen that has reacted with the magnetic particles from the surplus labeled antibody or labeled antigen and the liquid medium. (S10) The amount of excess labeled antibody or labeled antigen is measured by transmitted light (OD) and / or fluorescence intensity (FI). (S11) Excess labeled antibody or labeled antigen and liquid medium are discharged. (S12) Remove the measuring container from the magnetic field again. (S13) A dispersion liquid (for example, water containing a surfactant or physiological saline) is poured into the measurement container. (S14) After mixing, measurement is performed. Thus, the measurement of one sample is completed.

The washing liquid of the present invention is used during the above procedure, for example: (1) After the measurement of one sample is completed, when the measuring container is used again, the liquid adsorbed on the measuring container is washed. (2) After the sample is dispensed by the dispensing nozzle, the dispensing nozzle is washed by sucking and discharging the cleaning liquid by the dispensing nozzle. (3) Washing is performed by immersing the stirring probe after stirring the reaction solution in the measurement container in the washing solution. (A stirring operation with a stirring probe may be performed in the washing liquid). With the cleaning liquid of the present invention, components that affect other measurements can be effectively removed.

[0017]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples unless it exceeds the gist of the present invention.

Example 1 A fully automatic immunoassay system LPIA-A700 (manufactured by Mitsubishi Chemical Corporation) was used for measurement. As an immunizing reagent, a high-level HBs antigen sample (600000 IU / ml) was measured using a hepatitis B surface antigen measurement reagent LPIA-F.HBs antigen test (manufactured by Diatron), and washed with an alkaline washing solution having the following composition. Further, the fluorescence intensity of normal human serum (HBs antigen-negative) was measured using a measurement container washed with water (measured value A). On the other hand, normal human serum (HBs antigen-negative) was measured after measuring normal human serum (HBs antigen-negative) instead of high-value HBs antigen antibody, and then washing with an alkali washing solution and further using water. ) Was measured (measured value B). Measurement A and Measurement B
And the degree of the amount of HBs antigen derived from the high-value HBs antigen specimen adhered to the measurement container and remaining due to insufficient washing was calculated as the contamination rate of the following formula (I).

[0019]

(Equation 1)

As an alkaline washing liquid, a 0.25 N aqueous sodium hydroxide solution prepared with purified water is used, and as a surfactant (a) a polyoxyethylene alkyl ether nonionic surfactant (R = C ₁₈ H ₃₇ , n = 20, HL
B value 13.4) [product name: Brij7, manufactured by SIGMA
8] 0.05% by weight and (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₈ H
₁₇ , n = 9-10, HLB value 13.5) [manufactured by SIGMA, trade name Triton X-100] 0.05% by weight
Was used. The contamination rate is shown in Table 2.

Example 2 The surfactant to be added to the alkaline cleaning solution was (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₉ H ₁₉ , n = 20, HLB value 20.0) [Japan NP-2, manufactured by Surfactant Industries, Ltd.
0] 0.05% by weight and (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₈ H ₁₇ ,
n = 9 to 10, HLB value 13.5) [manufactured by SIGMA,
[Tradeon X-100] The contamination rate was measured in the same manner as in Example 1 except that 0.05% by weight was used. The results are shown in Table 2.

Example 3 The surfactant to be added to the alkaline cleaning solution was (a) a polyoxyethylene alkyl ether-based nonionic surfactant (R = C ₁₈ H ₃₇ , n = 20, HLB value 13.4) [SI
0.10% by weight of a brand name Brij78 manufactured by GMA and (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₈ H ₁₇ , n = 9 to 10, HLB)
Value 13.5) [SIGMA, product name Triton
X-100] The contamination rate was measured in the same manner as in Example 1 except that 0.10% by weight was used. The results are shown in Table 2.

Example 4 The surfactant to be added to the alkaline cleaning solution was (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₉ H ₁₉ , n = 20, HLB value 20.0) [Japan NP-2, manufactured by Surfactant Industries, Ltd.
0] 0.10% by weight and (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₈ H ₁₇ ,
n = 9 to 10, HLB value 13.5) [manufactured by SIGMA,
[Trade name of Triton X-100] The contamination rate was measured in the same manner as in Example 1 except that 0.10% by weight was used. The results are shown in Table 2.

Comparative Example 1 The surfactant to be added to the alkaline cleaning solution was (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₈ H ₁₇ , n = 9 to 10, HLB value 13.5)
[Product name: Triton X-100, manufactured by SIGMA]
The contamination rate was measured in the same manner as in Example 1 except that only one kind was changed to 0.10% by weight. The results are shown in Table 2.

Comparative Example 2 The surfactant to be added to the alkali cleaning solution was (a) a polyoxyethylene alkyl ether-based nonionic surfactant (R = C ₁₈ H ₃₇ , n = 20, HLB value 13.4) [SI
GMA, brand name Brij78] 0.1
The contamination rate was measured in the same manner as in Example 1 except that the amount was set to 0% by weight. The results are shown in Table 2.

Comparative Example 3 The surfactant to be added to the alkaline cleaning solution was (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₉ H ₁₉ , n = 20, HLB value 20.0) [Japan NP-2, manufactured by Surfactant Industries, Ltd.
0] The contamination rate was measured in the same manner as in Example 1 except that only one kind was changed to 0.10% by weight. The results are shown in Table 2.

Comparative Example 4 The surfactant to be added to the alkaline cleaning solution was (b) a polyoxyethylene aryl ether-based nonionic surfactant (R = C ₈ H ₁₇ , n = 9 to 10, HLB value 13.5)
[Product name: Triton X-100, manufactured by SIGMA]
The contamination rate was measured in the same manner as in Example 1 except that only one kind was changed to 0.20% by weight. The results are shown in Table 2.

[0028]

[Table 2]

As is clear from Table 2, the surfactant was 1
It can be seen that a mixture of a plurality of types has a lower contamination rate and a higher cleaning effect than a mixture of the types alone.

[0030]

By using the alkaline washing solution of the present invention in an automatic immunoassay apparatus, the washing effect can be remarkably improved, and a measurement with higher sensitivity becomes possible.

Claims (3)

[Claims] 1. An alkaline washing solution for an automatic immunoassay, comprising an alkaline aqueous solution containing two or more surfactants selected from the following (a) to (c). (A) Polyoxyalkylene alkyl ether-based nonionic surfactant (b) Polyoxyalkylene aryl ether-based nonionic surfactant (c) Alkyl diallyl ether sulfonate-based anionic surfactant 2. The surfactant according to claim 1, wherein the two surfactants selected from the above (a) to (c) are mixed at a weight ratio of 1: 9 to 9: 1. Alkaline washing solution for automatic immunoassay. 3. An immunoassay method using the alkaline washing solution according to claim 1 or 2 in a washing step of an automatic immunoassay method for measuring an antigen or an antibody in a sample by an antigen-antibody reaction.