JPH02161357A - Method for stabilizing material for immunosassay - Google Patents

Abstract

PURPOSE:To decrease the deactivation of antibodies and to allow the long-term preservation of the antibodies by cleaning a carrier combined with the antibodies with a cleaning liquid contg. a stabilizer. CONSTITUTION:The carrier combined with, for example, polyclonal antibodies, monoclonal antibodies, etc., is cleaned with the cleaning liquid contg. the stabi lizer and is then freeze-dried. The cleaning in this case merely involves immers ing of the carrier into the cleaning liquid and separating the liquid and the carrier and, therefore, the smaller amt. of the stabilizer to be used is necessitat ed. The freeze drying is executed by freezing the material for 2 to 24 hours in an incubator kept at, for example, -80 to -20 deg.C in an ordinary manner, then drying the material for 3 to 16 hours at -20 to 0 deg.C under 0.01 to 0.2Torr reduced pressure and further, drying the material for 2 to 4 hours at room temp. monosaccharides, such as glucose and fructose, disaccharides, such as cane sugar and lactic sugar, or dextran, polyvinyl pyrrolidone, etc., are used as the stabilizer. A physiological salt soln., a buffer soln. having pH to avert the denaturation of the combined antibodies, etc., are used.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for stabilizing a carrier to which an antibody is bound for use in immunological aPI determination. More specifically, the present invention relates to a method for stabilizing a carrier bound to an antibody used in immunoassay for a longer period of time and preventing deterioration of the antibody or the like.

(Prior art and its problems) In general, in order to measure the content of trace amounts of biological substances, such as proteins and hormones, that function as antigens in immune reactions in biological sample fluids such as serum and urine, antibodies are added to m-body. Immunoassay materials are often used that are combined with

Such immunoassay materials are often stabilized and stored in an appropriate buffer containing a protein such as bovine serum albumin. However, with such stabilization methods, there has been a problem in maintaining performance over a relatively long period of time, for example, until the immune reaction measurement material is delivered from the manufacturer to the user. That is, over a long period of time, for example, the antibody may peel off from the carrier, or the performance of the antibody itself (specific affinity performance with the antigen, etc.) may deteriorate.

Another stabilization method is known, which involves freeze-drying, but with this method, the antibody is likely to be deactivated during the freeze-drying operation, and slight differences in operating conditions may affect the degree of stabilization. Since changes occur in the freeze-drying process, cumbersome operations such as setting the conditions for the freeze-drying operation prior to operation and monitoring the conditions during the operation are required.

Under these circumstances, in the above-mentioned stabilization method by freeze-drying, there is also a method of freeze-drying the Immune Reaction A-1 regular use material together with a phosphate buffer containing a protein such as bovine serum albumin as a stabilizing agent. Also known. but,
In this method, white powder derived from proteins such as bovine serum albumin coexists in the immunoreaction 71$1 material produced, resulting in an unsightly appearance. Because it is difficult to dissolve, it requires some preparation time before use, and if an unexpected situation occurs during transportation of the material, the dried and powdered protein may easily scatter. There are challenges.

In view of the above-mentioned current situation, the present inventors developed a freeze-dried material that has less deactivation of antibodies bound to carriers, can be stored for a long period of time, and can be easily dissolved before use. As a result of intensive research, the present invention was completed. That is, the present invention provides a method for stabilizing a material for immunoassay, which comprises washing a carrier bound with an antibody with a washing solution containing a stabilizer and then freeze-drying the carrier.

The invention will be discussed in detail below.

(Means for Solving the Problems) In the present invention, antibodies refer to various antibodies obtained by known methods. Examples include polyclonal antibodies obtained by immunizing animals with antigens, and monoclonal antibodies produced by fused cells (hybridomas) of antibody-producing cells and dividing cells.

There are no equivalent restrictions on antibodies, such as their class, subclass, or origin.

The carrier may be any carrier as long as it can provide a surface for binding the antibody.

Methods for binding antibodies and carriers include methods that utilize chemical bonding and methods that utilize the adsorption power of the carrier. In this method, a carrier such as polystyrene that has an affinity with the antibody may be used, or a carrier whose surface is coated with a resin or the like that has an affinity with the antibody may be used. Further, the carrier may contain, for example, magnetic particles, and there are no restrictions on its size or the like.

The immunoassay material as used herein refers to the above-mentioned carrier itself or an immunoreaction container containing the above-mentioned carrier. Specifically, there are carriers that themselves provide a surface to which antibodies are bound, such as immunoreaction plates, and are also a material for immunoreaction.

Another specific form is a reaction cup that has beads (herein referred to as a carrier) therein that have antibodies bound to their surfaces.

The stabilization method of the present invention consists of washing the antibody-bound carrier with a washing solution containing a stabilizing agent, and then lyophilizing the carrier. Here, washing may be carried out, for example, by immersing the l'11 body in a washing liquid and separating it from the drained liquid. Therefore, the amount of stabilizer used in the present invention is small;
The cleaning operation can also be easily performed. Immunity/l
If the material used for regular use is itself a carrier, such as a plate, the material may be washed and freeze-dried; if the material consists of a carrier and a container containing the carrier, tu If the body is washed separately or the carrier is housed in a container or the like and then washed separately, they may be combined and freeze-dried. Freeze-drying may be performed under known conditions, for example, after freezing the material in an incubator at -80 to -20°C for 2 to 24 hours,
1~Q, 3~16 at -20~Q℃ under reduced pressure of 2 torr
An example is a method of drying for an hour and then drying for 2 to 4 hours at room temperature.

In the present invention, for example, monosaccharides such as glucose and fructose, dimonosaccharides such as sucrose and lactose, dextran, and polyvinylpyrrolidone can be used as stabilizers.

The washing solution can be used without particular limitation, such as physiological saline or a buffer solution with a pH level that does not denature the bound antibody.

The freeze-dried material obtained according to the method of the present invention can be packed, for example, in a state filled with dry air or nitrogen gas.
Furthermore, it can be stored stably for a long period of time.

(Effects of the Invention) According to the present invention, an immune reaction material can be stabilized for a long period of time. This means that the bound carrier is prevented from deteriorating and can be stored without reducing the performance of the material.

In the method of the present invention, most of the stabilizer, which is conventionally freeze-dried together with the carrier, is recovered together with the washing solution, so that the amount used during stabilization can be reduced. In addition, by collecting the washing liquid after washing in this way, the amount of water in the material can be reduced compared to the conventional method, and therefore the time required for the freeze-drying operation itself can be shortened. In addition, since the amount of stabilizer used can be reduced, unexpected problems can occur during transportation of manufactured materials! Even if the 11th state occurs, the amount of scattering of the stabilizer can be reduced.

Furthermore, the immunoreaction material stabilized according to the method of the present invention has good stability against heat, and under the conditions shown in the example, there is only a slight amount of deterioration even when it is left at 50 ° C. for 360 hours. .

Furthermore, when a monosaccharide, disaccharide, dextran, polyvinylpyrrolidone, or the like is used as a stabilizer, the material can be dissolved between supports when used.

Furthermore, by using sugars as stabilizers, the appearance of the manufactured material can be improved.

By packaging the freeze-dried material after filling it with dry air or nitrogen gas, the immunoreactive material can be stored for a longer period of time.

(Examples) Examples are shown below to explain the present invention in more detail, but the present invention is not limited to these Examples.

Example 1 (Stabilization of material for enzyme immunoassay of human α-fetoprotein) The solutions used in this example were: Solution A: 50mM sodium carbonate buffer (pH 9,6) Solution B: pH 7, with the following composition: 4 buffer sodium chloride - 5 g/l.

Potassium hydrogen phosphate - 0.2 g/l.

Disodium hydrogen phosphate - 1.15 g/l Potassium chloride - 0.2 g/l tween 20 - 0.5 ml/1 Prepared according to the usual method in each well of a polystyrene microtiter plate (Immunoplate ■ manufactured by Nunc). 5 μg/ml of a mouse monoclonal antibody against human α-fetoprotein (hereinafter referred to as AFP) was added to 2
00 ul was dispensed.

At this time, solution A was used as an antibody dissolution solution.

After leaving the plate at 4°C overnight, it was washed with solution B, and for blocking, 0.1% bovine serum albumin was added.
300 u of B was added to each well to give a nail opening %.
The mixture was dispensed into 1-liter portions and left overnight at 4°C.

After each well was washed with a 0.85% by weight aqueous sodium chloride solution and further washed with physiological saline containing sucrose heated 10 times, the drained fluid was removed and the plate was freeze-dried according to a conventional method. .

As a control, a similarly treated plate that had not been freeze-dried was used.

For freeze-dried plates, each well in the plate was filled with Solution B, which was removed and used for the following measurements.

For API constant, add 20ul of AFP antigen solution (0,
6,25,12,5,25,50,100°200.4
After adding 300ul of anti-AFP mouse antibody labeled with horseradish peroxidase, leave it at room temperature for 1 hour, and add solution B. Wash 3 times and add peroxidase substrate solution (ABTS and hydrogen peroxide solution)
After 200 ul was added and left to stand for 30 minutes, 100 ul of sychoic acid solution (pH 1,0) was added to stop the reaction, and the absorbance at 415 nm of each well was measured.

FIG. 1 shows the results of a control example and a plate prepared by the method of the present invention subjected to freeze-drying.

Comparative Example 1 A plate prepared in the same manner as the freeze-dried plate of Example 1 except that the wells were washed with sucrose-free physiological saline before freeze-drying was treated in the same manner as in Example 1. 891 was determined.

The results are also shown in Figure 1.

Example 2 A plate prepared in the same manner as in Example 1 was freeze-dried, then pouched in an aluminum bag (plain aluminum bag 170 x 240 pieces manufactured by Tofujibag ■) and incubated at 50°C for 360 hours. In addition, as a control, a plate prepared in the same manner but not freeze-dried, pouched in the same manner as in Example 2, and incubated at 4°C for 360 hours was used.

Measurements were performed on these plates in the same manner as in Example 1. The results are shown in Figure 2. For freeze-dried plates, each well is filled with B solution and removed prior to measurement.

Comparative Example 2 Example 2 was performed on a plate prepared in the same manner as the freeze-1+'i-dried plate of Example 1, except that the wells were washed with sucrose-free physiological saline before freeze-drying. After pouching and incubating at 50°C for 360 hours, measurement was performed. The results are also shown in Figure 2.

Example 3 A human AFP rabbit antibody was bound to a microtiter plate in the same manner as in Example 1, and blocking was performed in the same manner as in Example 1. After washing the plate three times with physiological saline, it was washed once with physiological saline containing the reagents shown in Table 1 to remove the wetted liquid. After freeze-drying this plate, it was pouched in the same manner as in Example 2 in the presence of nitrogen gas.

The plate prepared as above was heated to -20°C or 50°C.
The cells were left in an incubator for 120 hours. After filling each well in the plate with solution B and removing it,
Measurements similar to those in Example 1 were performed using AFP antigen 'ttl at a concentration of 0 ng/ml.

The results are shown in Table 1 (however, the results in Table 1 are
It shows the value obtained by subtracting the absorbance at 415 nm when no antigen solution was added from the absorbance when 400 ng/mNa degree antigen solution was added).

Table 1 However, BSA stands for bovine serum albumin, PEG stands for polyethylene glycol, and pvp stands for polyvinylpyrrolidone.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of Example 1 and Comparative Example 1, and FIG. 2 is a diagram showing the results of Example 2 and Comparative Example 2. In both figures, (b) shows the control in each example, (○) shows the results when using the plate prepared according to the method of the present invention in each example, and (△) shows the results of each comparative example. Each is shown below.

Claims (4)

[Claims] (1) A method for stabilizing a material for immunoassay, which comprises washing a carrier bound with an antibody with a washing solution containing a stabilizer and then freeze-drying it. (2) The method according to item (1), wherein the stabilizer is at least one selected from monosaccharides, disaccharides, dextran, and polyvinylpyrrolidone. (3) The method according to claim (1) or (2), wherein the carrier is an immunoreaction plate. (4) A method for stabilizing an immunoassay material, which comprises filling the immunoreaction material obtained by the method according to claim (1) with dry air or nitrogen gas and packaging it.