JPS59206761A - Enzyme immunomeasuring method - Google Patents

Abstract

PURPOSE:To prevent deactivation of an antigen, antibody and enzyme owing to drying-up and to enable always exact immunomeasurement by treating the surface of an enzyme marked antibody sensitized on a carrier or the same marked antigen or non-marked antibody or antigen with protein to protect said surface. CONSTITUTION:The sensitized surface of a carrier such as a microplate or the like sensitized with an antibody marked with enzyme such as peroxidase or the like or the similarly marked antigen or non-marked antibody or antigen in each well of the carrier is put therein with a buffer soln. contg. gelatin, albumin of a cattle serum, egg white, etc. and after resting for a several seconds, the injected liquid is adsorbed away and the carrier is rested to dry at about 25 deg.C. A protective film of protein is thus formed on the surface of the sensitized wells and the always exact immunomeasurement is accomplished without deactivation of the antigen, antibody and enzyme deposited on the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to enzyme immunoassay, which is one of immunochemical assay methods.
Hereinafter, it is related to [3IA].

Immunochemical assay methods for measuring various proteins, hormones, etc. that act on living bodies with high sensitivity have recently been gaining importance in the field of clinical testing. EIA is known as one of the immunochemical measurement methods, and in this measurement method, for example, an antigen or antibody is labeled with an enzyme such as peroxidase,
It is marketed by a method that determines the reactivity (enzyme activity) between this and the substrate. E [Due to differences in measurement methods, A is the Sanderutsch method,
It is further divided into two antibody methods, competitive methods, etc., but the basic principle is that antibodies and antigens are insolubilized in a suitable carrier, and non-analyte substances such as antigens and antibodies are allowed to act on this, and the amount thereof is measured. For this purpose, antibodies and antigens are labeled with an enzyme, then reacted, and then separated into the same phase and liquid phase, and the enzyme activity value of the same phase or liquid phase is measured. They have the same thing in common: knowing the value of the target substance.

Therefore, during the measurement of E[Δ, it is important to ensure that the activity of the antigen-antibody insolubilized in the carrier and the enzyme activity of the enzyme-labeled antibody or the same antigen are not deactivated.

Recently, in the field of clinical test reagents, it is necessary to quickly process five types of specimens, so we have developed a so-called microplay system that can be easily automated using certain equipment. There is a need to develop a method using ~ as a carrier.

However, when using a microplate as a carrier, it is necessary to wash the plate during measurement and dry the well surface of the carrier when performing a dispensing operation, but this drying causes the bonding to the well surface of the carrier. There is a drawback that the activity of the enzyme-labeled antibody or the same antigen or the activity of the antibody or antigen is reduced. Therefore, if these activities decrease during the drying stage, accurate measured values cannot be obtained.

In view of the above circumstances, the present inventors have developed a method for suppressing the deactivation of the activity of the sensitizer on the carrier during the measurement, especially during the drying stage of the carrier, when conducting measurement tests using conventionally known ETA. As a result of various studies to achieve this goal, we have discovered that the object of the present invention can be achieved by treating the enzyme-labeled antibody or the same antigen or the surface of the carrier to which the antibody or antigen is bound with a certain substance, and have completed the present invention. 1 [.

That is, the gist of the present invention is that in enzyme immunoassay,
It consists in an enzyme-labeled antibody or antigen sensitized on a carrier, and an enzyme immunoassay method characterized in that the surface of the antibody or antigen is treated with a protective agent made of protein.

The present invention will be explained in detail below.

Although the present invention is directed to a measurement method using EIA, it can be applied to any method such as the Sand-Deutsch method, the dual-antibody method, or the competitive method.

Further, in the present invention, the target substances whose activity reduction can be suppressed using these known measurement methods include the enzyme activity of an enzyme-labeled antibody or antigen, and the activity of the antibody or antigen.

The carrier for insolubilizing proteins such as antibodies and antigens is not particularly limited, but in the present invention, when a microplate having a large number of wells is used,
This is preferable because it produces a greater effect. The material of the carrier is determined by taking into account nonspecific adsorption amount, immobilization capacity, chemical stability, material uniformity, etc. depending on the properties of the various substances. For example, synthetic resins such as polystyrene and polycarbonate are suitable. be.

Examples of the enzyme for labeling used in the present invention include peroxidase, β-D-galactosidase, alkaline phosphatase, etc. Among them, peroxidase can be used most widely due to its characteristics. Therefore, it is preferable.

In the present invention, it is essential to treat the surface of the carrier with a protective agent made of protein in order to suppress the activity of the enzyme-labeled antibody or antigen bound to the carrier, or the antibody or antigen. be. Specific examples of this protein include egg white, bovine serum albumin (hereinafter referred to as B
, S, A) or gelatin. Note that hydrolyzed gelatin can also be used in the same manner.

The protective agent is usually applied onto the carrier by adding 0.01 to 5% by weight, preferably 0.05 to 2% by weight of the measuring reagent, such as a washing solution used for separation of bound and unbound substances (B/F separation). This can be easily carried out by adding % of the above-mentioned protective agent and washing the carrier with this solution. This washing is usually carried out by bringing the solution into contact with the well surface of the carrier for about 1 to 60 seconds.

In the present invention, treatment with this protective agent suppresses deactivation of enzyme-labeled antibodies or antigens bound to the carrier due to drying.

The cause of this is not clear, but molecules of the protective substance rapidly adsorb onto the well surface of the carrier, coating and protecting the surface of the sensitized material that has been adsorbed to the well surface first, resulting in rapid drying. It is thought that trifles that become inactive depending on the conditions are suppressed.

In the present invention, the carrier that has been treated with the above-mentioned protective agent is then further treated in a known measurement order according to a conventional method.
Ill be beaten. For example, sensitize an enzyme-labeled antibody and apply a protective agent thereon: lI! After drying the carrier, the enzyme activity can be measured by reacting it with an enzyme substrate.

As described above, the present invention suppresses deactivation of a sensitizer such as an enzyme-labeled antibody bound to a carrier during drying, so it is particularly suitable for a measurement method using a microplate as a carrier, and allows for more accurate measurements. You can expect it. Further, in the present invention, coating the surface with a protective agent does not have any adverse effect on the measured values or other disadvantages.

Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples unless the gist thereof is exceeded.

Examples 1 to 3 and Comparative Examples 1 to 4 An enzyme-labeled antibody solution containing peroxidase was injected with a pipette into each well (volume 0, 35 m!; L) of a carrier made of a polystyrene microplate, and
After allowing the solution in each well to stand overnight in the RI bath of C, the solution in each well was absorbed and removed using a bibet 1~. (!I 7) Next, in each well of this carrier, a solution containing 1% of the protective agent shown in Table 1, pH 8.0, was poured and injected with a pipette, and after 5 seconds, the solution was poured again. The main gyoza solution was absorbed and removed.

Micro play with such processing! - then
After drying by leaving at a temperature of 25 DEG C. for the time shown in Table 1, the enzyme substrate was injected into each wool at the same rate, and the enzyme activity was measured by fermentation reaction at room temperature for a certain period of time. In addition, the measurement of alcoholic ζold↑1゜ (peroxydavi activity) was performed using ABl-8 (2,2'--Azine Bis(3.Cobourg\Penzothiaso"Lin.6.Sulol)) as the substrate. was used at 10 mM, dispensed 0.3111 μl into each well, and incubated at room temperature (2
5°C) for 30 minutes and then measured.

Then, when the enzyme activity without drying is taken as 100%, the reduction in activity due to the drying treatment υ] was determined as a relative value, and the results are shown in Table 1.

Table 1 From the results shown in Table 1, when the protective agent of the present invention was added to +311 Schiffco solution and treated, the dry trough of the carrier was 8.
It can be seen that even if the enzyme activity is longer than 60 minutes without drying, there is not much difference between the enzyme method and the raw enzyme method. I understand.

Examples 4 to 6 and Comparative Example 5 In the method of Example 1, P B S 6"c '71
'The method was exactly the same except that the surface of the well was treated with the addition of 0.1% of the protective agent shown in Table 52'. The results are shown in.

Table 2 From the results in Table 2, it is clear that the present invention has 11 (MaQ, 1%)
Even in extremely small amounts, the enzyme activity is sufficiently effective to prevent deactivation.
I.

° Examples 7 to 9 and Comparative Example 6 The method of Example 1 was exactly the same except that the gratin water level shown in Table 3 was used, and the microplate was dried rapidly by blowing air instead of being left to dry. When measurements were carried out using this method, the results were shown in Table 3.

From the results shown in Table 3, the effect of the protective agent of the present invention is that even in the case of rapid drying treatment by air blowing, a decrease in enzyme activity can be significantly prevented with a small amount added.

Examples 10 to 12 and Comparative Example 7 First, a microplate was sensitized with antibodies.The sensitization method was the same as the sensitization method for enzyme-labeled antibodies, and antibodies were added to PB at arbitrary concentrations.
The solution was diluted with S solution, dispensed into tubes, and allowed to stand overnight at 4°C.Then, the solution was removed by suction, and the antibody-sensitized carrier was added to 1"I.

The antibody used was anti-goat cancer macropurin G antibody.

Then, as before, PB containing each protective agent <1 wt%)
S solution (control was untreated), followed by drying at room temperature (25°C for 11 hours), freeze-drying, followed by treatment to prevent non-specific binding of the enzyme-labeled antibody, and then optionally Add 1 liter of enzyme-labeled goat comma globulin G antibody diluted in 3S solution to 0°3°C.
The culm antigen-antibody reaction was carried out at 37°C for 2 hours, then washed with PBS solution, and finally the same substrate (ABT
0.3n+ of S> was dispensed into each well, and an enzymatic reaction was performed. In other words, the activity of the sensitized antibody can be indirectly verified by measuring the amount of enzyme activity of enzyme-labeled goat gamma globulin G captured by the sensitized antibody. The activity is calculated as a relative value with the non-dried case as 100%, and the fourth
The results shown in the table were obtained.

From the results shown in Table 4, it can be seen that the protective agent of the present invention has the effect of suppressing the decrease in activity not only of enzyme-labeled antibodies but also of sensitized antibodies.

Agent: Patent Attorney Tsutomu Setatetsu 1 idiot

Claims (1)

[Scope of Claims] 1. An enzyme-labeled antibody sensitized on a carrier, or the antigen, or the surface of the antibody or antigen, is treated with a protective agent made of protein in the enzyme-linked immunoassay. . 2. The enzyme immunoassay method according to claim 1, wherein the protective agent is egg white, bovine serum albumin, or gratin. 3. The enzyme immunoassay method according to claim 1, wherein the treatment with the protective agent is a method of immersing the carrier surface in a measurement reagent solution containing 0.01 to 5 weight of the protective agent. . 4. The enzyme immunoassay method according to claim 1, wherein the carrier is a microplate. 5. The enzyme immunoassay method according to claim 1, wherein the material of the carrier is a synthetic resin. 6. The enzyme immunoassay method according to claim 1, wherein the enzyme that forms the enzyme-labeled antibody or the antigen is peroxidase.