JPH05264549A - Enzyme immunoassay method - Google Patents

Abstract

PURPOSE:To achieve a highly accurate measurement easily and quickly by preventing a drop in the amount of chemiluminescence after the washing of a solid phase by using a tris buffer liquid as washing liquid in the measurement of horseradish peroxidases (HRP) by a chemiluminescence method washing a solid phase antibody. CONSTITUTION:In an enzyme immunoassay method in which a solid phase antibody is caused to react with an object to be measured and after a reaction with HRP as labeled enzyme, the solid phase antibody is washed to perform a measurement of the HRP by a chemiluminescence method, a tris buffer liquid is used as washing liquid for washing. The object to be measured are colibusilli and a tris-hydrochloric acid buffer is used as tris buffer to perform an enzyme immunoassay by a sandwiching method. A measurement of the HRP is performed by an intensified chemiluminescence method using P-iodophenol as intensifier. This enables the checking for the effect of measuring errors due to time during which the solid phase antibody alone thereby achieving higher measuring accuracy of microorganism.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an enzyme immunoassay method,
In particular, it relates to an enzyme immunoassay method using horseradish peroxidase (HRP) as a labeling enzyme.

[0002]

2. Description of the Related Art Conventionally, an enzyme immunoassay method in which EIA is carried out using HRP as a labeling enzyme has been used for the measurement of Escherichia coli and the like, and many such measurement methods have been reported.

In this method, the solid phase is washed with a washing solution under various conditions before the enzymatic reaction, and in particular, distilled water, physiological saline, buffer solution or the like is usually used as the washing solution. ..

In the EIA, after the second reaction, the solid phase is washed to carry out an enzymatic reaction. When the measurement is performed by the chemiluminescence method, the enzymatic reaction is a luminescent reaction. Therefore, when the luminescence measurement is performed for each specimen, many samples are left in a dried state until the luminescence measurement after washing.

[0005]

However, when the solid phase is washed with distilled water or the like after the second reaction as described above and the sample is allowed to stand in a dried state until luminescence measurement, as shown in FIG. As the temperature becomes longer, the amount of chemiluminescence decreases.

This phenomenon causes a large decrease in measurement accuracy when a calibration curve is prepared using a standard substance having a known concentration and the concentration of the sample to be measured is obtained from the chemiluminescence amount, which is a major obstacle.

Therefore, when a large number of specimens are measured, washing cannot be performed collectively, and it is necessary to repeat washing and measurement for each specimen, and such a very complicated operation is performed every measurement. I had no choice.

The present invention has been made under the background described above, and provides an enzyme immunoassay method which prevents the decrease in chemiluminescence amount after solid phase washing as described above, is easy and rapid, and is highly accurate. The purpose is to

[0009]

Means and Actions for Solving the Problems In order to solve the above problems, the present invention reacts a solid-phase antibody with a measurement target, and then reacts this solid-phase antibody with a labeling enzyme, horseradish peroxidase (HRP). In the enzyme immunoassay method in which the solid phase antibody is washed and then HRP is measured by a chemiluminescence method, a Tris buffer is used as a washing solution when the washing is performed.

In the enzyme immunoassay described above, the measurement target is Escherichia coli, a Tris-hydrochloric acid buffer is used as the Tris buffer, and the enzyme immunoassay is performed by a sandwich method. An enzyme immunoassay method is also provided, wherein the measurement is performed by an enhanced chemiluminescence method using P-iodophenol as a sensitizer.

By washing the solid phase antibody with Tris buffer as described above, it is possible to suppress the influence of the measurement error due to the standing time of the solid phase antibody and increase the measurement accuracy of the microorganism.

In the above-mentioned enzyme immunoassay, microorganisms and the like can be mentioned as the object of measurement, preferably coliforms, more preferably ultrasonically disrupted coliforms.
The enzyme immunoassay is preferably a non-competitive method, more preferably a sandwich method.

The enhanced chemiluminescence method is preferred as the method for measuring horseradish peroxidase, and more preferably P-iodophenol is used as the sensitizer.

The material of the solid phase is not particularly limited, but a plastic test tube or the like is preferably used. In the present specification, a solid phase coated with an antibody, for example, an anti-coliform IgG IgG-coated polystyrene test tube is referred to as a solid phase antibody.

[0015]

Example In this example, coliforms were selected as the microorganisms to be measured, and HRP-labeled rabbit anti-coliform F was added to the coliforms.
The coliforms were measured by using Tris-hydrochloric acid buffer as a washing liquid after the reaction after adding ab '. The enzyme immunoassay method for coliform bacteria is shown below.

A, enzyme immunoassay (1) 0.1 ml / l containing 0.1 ml of ultrasonically disrupted coliforms, 0.1% bovine serum albumin and 0.1% sodium azide
0.2 ml of borate buffer (pH 8.5) was added to rabbit anti-coliform IgG
Place in a coated polystyrene test tube.

(2) After reacting at room temperature for 1 hour, the solid phase is washed 5 times with distilled water. (First wash) (3) Add 0.3 ml of HRP-labeled rabbit anti-coliform Fab '.

(4) After reacting at room temperature for 1 hour, the solid phase is washed. (Second cleaning) (5) 1.2 × 10 ^-5 mol / l luminol, 4 × 10 ^-4 mol hydrogen peroxide, 1.82 × 10 ^-4 mol P-iodophenol, 0.1m
Add 1 ml of ol / l Tris-HCl buffer (pH 8.5).

(6) The amount of chemiluminescence for 16 to 45 seconds is measured.

In this embodiment, the second washing is performed four times with distilled water and then 0.1 mol / l Tris-HCl buffer (pH) is added.
It is performed by washing once with 8.5), and the leaving time is 0
The amount of chemiluminescence was measured after 180 minutes. This is cleaning example 1
And

In addition, as a comparative example, after the second washing was performed 5 times with distilled water, the leaving time was 0 to 18 as in the washing example 1.
The amount of chemiluminescence was measured as 0 hour. This is designated as Cleaning Example 2.

The measurement results of the chemiluminescence amount of the cleaning examples 1 and 2 are shown in FIGS.

[0023]

[Table 1]

In Table 1, the chemiluminescence amount at each measurement point was expressed as a percentage with the chemiluminescence amount at 0 minutes of standing time being 100%, and this was expressed as a retention rate (%).

[0025]

## EQU1 ## Retention rate = 100 × (amount of chemiluminescence at standing time x minutes / amount of chemiluminescence at standing time 0 minutes) In Table 1, each chemiluminescence amount is an average value of 5 repeated measurements. The coefficient of variation CV (%) is 3.4 to 12.
It was 9%.

Lines A1 to A6 in FIG. 1 are graphs of the amount of luminescence with respect to the number of coliform bacteria at the standing times of 0, 30, 60, 90, 120 and 180 minutes, respectively. Similarly, lines B1 to B6 in FIG.
The graph of the luminescence amount with respect to the number of coliforms at 0, 60, 90, 120, and 180 minutes is shown.

As shown in Table 1 and FIG. 1, in Washing Example 1 in which the fifth washing was performed with Tris buffer, the calibration curve did not change so much even if the standing time was increased, and 180
The retention rate was 80 to 120% even after standing for a minute, and there was almost no effect of standing.

On the other hand, in the cleaning example 2 in which the second cleaning was performed only with distilled water, the amount of luminescence decreased with the passage of time, and as shown in FIG. The slope is also decreasing, and therefore the retention rate is also decreasing with time.

From the above results, it can be seen that the use of Tris buffer in the second washing can suppress the decrease in chemiluminescence amount due to standing after the washing.

[0030]

As described above, according to the enzyme immunoassay method of the present invention, when processing a large number of samples, highly accurate measurement can be performed regardless of the difference in the standing time of each sample.

Further, since it becomes possible to carry out the processing of the sample all at once, the operation becomes simple and the measuring time can be shortened.

[Brief description of drawings]

FIG. 1 is a graph showing the amount of chemiluminescence with respect to the number of coliform bacteria in the enzyme immunoassay method according to one example of the present invention.

FIG. 2 is a graph showing the chemiluminescence amount with respect to the number of coliform bacteria in the conventional enzyme immunoassay method.

Claims (2)

[Claims] 1. A solid phase antibody is reacted with an object to be measured, and then the solid phase antibody is reacted with a labeling enzyme, horseradish peroxidase (HRP), and then the solid phase antibody is washed and subjected to chemiluminescence method. In the enzyme immunoassay method for measuring HRP, a Tris buffer is used as a washing solution when the washing is performed. 2. The enzyme immunoassay method according to claim 1, wherein the measurement target is coliform bacteria, a Tris-hydrochloric acid buffer solution is used as the Tris buffer solution, and the enzyme immunoassay is performed by a sandwich method. The enzyme immunoassay method is characterized in that the HRP is measured by an enhanced chemiluminescence method using P-iodophenol as a sensitizer.