JPH1031020A - Measuring method using antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method, by which a specific analyzing substance can be measured in a short time without requiring any B/F separation, in an analyzing method based on an antibody. SOLUTION: Using a labeled antibody labeled with a labeling substance to a specific constituent, the specific constituent is measured. In this case, the labeled antibody is allowed to react with the specific constituent and an antibody-antigen complex is formed, and then, the labeling substance is analyzed without carrying out any B/F separation. Analysis of the labeling substance is inhibited by steric hindrance if the antibody-antigen complex is formed, while analysis of the labeling substance is not inhibited if the antibody-antigen complex is not formed, so that concentration of the specific constituent, whose concentration is unknown, can be found on the basis of a calibration curve, which is formed previously from the analysis result of the labeling substance and the concentration of the specific constituent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reaction for forming an antigen-antibody complex using an antibody and a specific component, by utilizing the specific binding property of the antibody, and to labeling the antibody with a label. The present invention relates to a method of labeling a substance, analyzing the labeled substance, and measuring a specific component from the analysis result. More specifically, the present invention relates to a method for determining the concentration of a specific component in a clinical test, without performing B / F separation, which is usually performed for measurement of a specific component using an immunoassay.

[0002]

2. Description of the Related Art When a specific component is measured using an antibody, a labeled antibody, which is labeled with a labeling substance and specifically binds to the specific component, is immobilized on an inner surface of a microplate or the like in advance, and the antigen is measured. After the primary reaction of the antibody reaction, B / F separation is performed, and a specific component is indirectly measured from a signal obtained by analyzing a labeling substance as a secondary reaction using a calibration curve prepared in advance.

[0003] However, when an antibody labeled with a labeling substance is immobilized, a 96-well microplate is often used, but the amount of the antibody immobilized is limited only by the inner surface of the microplate. Is limited. Since the antigen-antibody reaction is also performed only on the inner surface, the reaction must be performed for a long time to complete the reaction.

[0004] B / F separation is always required before analyzing the labeling substance as a secondary reaction. Since the B / F separation is performed using a cleaning liquid or a cleaning device, the burden on the measurer is large and it is not suitable for urgent measurement. Enzymes are often used as labeling substances, and the enzyme reaction has high detection sensitivity, but the reaction time is long, and the secondary reaction is performed only on the inner surface of the microplate, so the time until the end of measurement is long. Had been reached.

[0005]

SUMMARY OF THE INVENTION According to the present invention, an antigen-antibody reaction is performed without immobilizing a labeled antibody in an analysis method using an antibody, and no B / F separation is required for analyzing a labeled substance. Provided is a measurement method in which the analysis of a labeling substance is completed in a short time and the time from the start of measurement to the end of measurement can be reduced.

[0006]

According to a method for measuring a specific component using a labeled antibody which specifically binds to a specific component to be measured and is labeled with a plurality of detectable labeling substances, (1) After a specific component is reacted with a labeled antibody to form an antigen-antibody complex, the labeled substance is analyzed without performing B / F separation. (2) When analyzing a labeling substance, when an antigen-antibody complex is formed, the analysis of the labeling substance is hindered by steric hindrance. When no antigen-antibody complex is formed, the analysis of the labeling substance is hindered. The analysis result of the labeling substance is determined according to the amount of the antigen-antibody complex. (3) In order to obtain the measurement result of the specific component, the operations of (1) and (2) are performed using the specific component having a known concentration, and a calibration curve is previously determined from the analysis result of the labeling substance and the concentration of the specific component. A measurement method characterized in that a specific component concentration of a sample whose concentration is unknown is determined based on a calibration curve prepared beforehand.

[0007] Since a labeling substance for labeling an antibody is analyzed as a secondary reaction following the antigen-antibody reaction, it is necessary to react specifically and in a short time. The avidin-biotin reaction is completed immediately after the binding reaction, and the specificity is very high. Biotin labeling of an antibody is widely and generally performed. When a biotin labeling agent that reacts with an amino group in an antibody is used, about 10 to 20 biotins can be labeled. Since there is no need to increase the detection sensitivity by the enzymatic reaction, the analysis result can be obtained in a very short time.

[0008] For the purpose of obtaining an analysis result of the amount of labeled biotin, the above antibody is combined with avidin and 2- (4'-H).
hydroxyazobenzene) benzoic
An avidin-HABA solution to which acid (hereinafter referred to as HABA) is bound is used. Avidin-HABA solution
Avidin and HABA are bound by a weak force and have an orange color. In the presence of biotin, the reaction between avidin and biotin is stronger than the binding between avidin and HABA.
The absorbance at m decreases. Since the substitution of biotin and HABA occurs quantitatively, it is used for the quantification of biotin as the avidin-HABA method.

Next, the fact that avidin-HABA does not react with biotin due to steric hindrance when an antigen-antibody complex is formed is described below. In some cases, a plurality of antibodies may bind to one molecule of antigen, even if the antibody is a monoclonal antibody. For example, C-reactive protein (hereinafter C
RP) has a pentameric structure, and in the case of a monoclonal antibody, one antigen molecule has five antibody binding sites. Depending on the combination of monoclonal antibodies, more antibodies can be bound. By forming an antigen-antibody complex between the antigen which is the specific analyte and the antibody labeled with the labeling substance, even if the labeled biotin and avidin present on the surface of the antigen-antibody complex molecule can be bound Most of the biotin present in the antigen-antibody complex molecule is
Cannot bind to avidin due to steric hindrance. Therefore H
ABA and biotin substitution reactions are not performed
No decrease in absorbance was observed. Therefore, the relationship between the amount of antigen and the absorbance is low when the amount of antigen is low,
When the amount of antigen is large, the absorbance increases, and the calibration curve rises to the right according to the amount of antigen.

A reaction system in which the calibration curve rises to the right has one advantage in the measurement method. In general, such a competitive reaction is strong in a low concentration range and the absorbance is high,
When the concentration is high, the reaction becomes weak and the absorbance decreases, so that the calibration curve often falls to the right. In clinical test items, normal values are often in the low concentration range, and abnormal values are very often in the high concentration range.In the case of the calibration curve falling to the right, the numerical values of the measurement results in the low concentration range vary greatly. In other words, if the cutoff value that separates the normal value from the abnormal value is close to the normal value, the value may be determined to be an abnormal value due to a variation in the value, although the value is originally a normal value.

If the calibration curve rises to the right, the variation in the normal value in the low concentration range is small, and thus the influence on the accuracy of the measurement result is small. On the other hand, even if the variation in the abnormal value range is large, the abnormality is abnormal. It can be said that using a calibration curve that rises to the right is a very ideal measurement method because the value can be easily determined.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described by taking as an example a measurement performed using a biotin-labeled anti-CRP monoclonal antibody using CRP as a specific analyte. (Embodiment 1) Preparation of various solutions used in the present invention will be described below. (Preparation of biotin-labeled antibody) NHS-biotin (manufactured by Dojin Chemical Co.) was added to an anti-CRP antibody (manufactured by Rockland) solution, left at room temperature, and dialyzed using a dialysis membrane to remove unreacted biotin. I do.

(Preparation of HABA solution) To 9.8 ml of water was added 0.2 ml of 1N NaOH, and 24.2 mg of HA was added.
BA (manufactured by Nacalai Tesque) is dissolved to obtain an HABA solution.

(Preparation of PBS solution) A mixture of 10 mM sodium phosphate and 150 mM sodium chloride was adjusted to pH
Adjust to 7.2.

(Preparation of Avidin-HABA Solution)
8 mg of avidin (Sigma) in 4 ml of PBS solution
And 600 μl of the HABA solution.

(Preparation of CRP antigen standard solution) CRP antigen (manufactured by Chemicon) was added to PBS solution at 0 μg / m
1, 50 μg / ml, 100 μg / ml, 200 μg / ml
Prepare a 500 ml / ml CRP antigen standard solution.

(Embodiment 2) A calibration curve was prepared using the above-mentioned various adjustment solutions.

CRP antigen standard solutions of each concentration were
0 μl was collected, and 80 μl of a biotin-labeled antibody adjusted to 400 μg / ml was added and mixed to allow the antigen-antibody reaction to proceed.
Allow to run for 10 minutes in a hot water bath. Next, avidin-HAB
900 μl of the A solution is added, stirred, left at room temperature for 3 minutes, and the absorbance at 500 nm is measured using a spectrophotometer. FIG. 1 shows a calibration curve of CRP.

[0019]

As described above, the measurement method using the antibody of the present invention can provide a measurement result in a short time without the need for B / F separation. In addition, since it can sufficiently respond to an urgent need for a specific component concentration, it can be used for measurement of various specific components in the fields of biochemical analysis and clinical testing.

[0020]

[Brief description of the drawings]

FIG. 1 is a calibration curve of CRP in the embodiment.

Claims (3)

[Claims] 1. A method for measuring a specific component using a labeled antibody which specifically binds to a specific component to be measured and is labeled with a plurality of detectable labeling substances, comprising: After reacting the components with the labeled antibody to form an antigen-antibody complex, the labeled substance is analyzed without performing B / F separation. (2) When analyzing a labeling substance, when an antigen-antibody complex is formed, the analysis of the labeling substance is hindered by steric hindrance. When no antigen-antibody complex is formed, the analysis of the labeling substance is hindered. The analysis result of the labeling substance is determined according to the amount of the antigen-antibody complex. (3) In order to obtain the measurement result of the specific component, the operations of (1) and (2) are performed using the specific component having a known concentration, and a calibration curve is previously determined from the analysis result of the labeling substance and the concentration of the specific component. A measurement method characterized in that a specific component concentration of a sample whose concentration is unknown is determined based on a calibration curve prepared beforehand. 2. The method according to claim 1, wherein biotin is used as a labeling substance for labeling the antibody. 3. An avidin-HA for analyzing a labeled substance.
The method according to claim 2, wherein a BA solution is used.