JPS6266141A - Vessel for fluorescent immunological measurement - Google Patents

Abstract

PURPOSE:To measure fluorescence with high accuracy by providing a layer to reflect the fluorescence to a vessel for contg. the sample which emits the fluorescence, onto the surface not in contact with the sample. CONSTITUTION:The layer 2 which consists of a metal or contains the metal and reflects the fluorescence is provided to the outside surface of a plastic molding 1 for contg. the sample which emits the fluorescence. Since the fluorescence emitted by the sample is reflected by the layer 2, the fluorescent intensity sensed by a measuring instrument is increased and the measurement in a trace region is made possible with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a sample container used in fluorescence immunoassay, which is a method of immunoassay.

[Prior art]

Fluorescence analysis is widely used as a method for ultra-trace analysis, and today, fluorescence immunoassay, an immunoassay that applies this measurement technology, is being used in clinical testing.

This measurement method is a highly sensitive immunoassay developed from a method commonly called enzyme immunoassay, and is capable of measuring trace amounts that exceed radioimmunoassay, which was said to be the most sensitive method up until now. It is said that there is. In this measurement method, an antigen (or antibody) is adsorbed and immobilized on the inner surface of a container, then the antibody (or antigen) is added to cause an antigen-antibody reaction to occur sequentially, and finally the enzyme-labeled antibody (or antigen) is added. Make it react. Furthermore, a fluorescent substrate (a substance that emits fluorescence when the enzyme reacts) is added to the enzyme immobilized on the sample, and excitation light is irradiated onto it, and the amount of fluorescence emitted by the sample is read.

JP-A-59-132335 has been known as a container for use in fluorescence immunoassay.

This is an attempt to accurately detect the fluorescence emitted by the sample by suppressing the self-fluorescence emitted by the material that makes up the container itself. Specifically, pigments are kneaded into the plastic material that makes up the container. Or coated 1. This is an attempt to reduce the occurrence of self-fluorescence by preventing excitation light from penetrating into the molded product. However, as disclosed in Japanese Patent Application Laid-Open No. 60-6868,
When additives such as pigments are added to plastic materials, there is a problem that non-uniformity occurs in the adsorption of antigens (or antibodies), etc. on the surface of the plastic materials. It cannot be said that it is a thing.

On the other hand, there are many low-fluorescence materials on the market that have low self-fluorescence and have little effect on actual fluorescence measurements, and measurement equipment is also difficult to determine whether the excitation wavelength or the fluorescence wavelength. It has also been improved to automatically correct for self-fluorescence, which causes pack-ground. Therefore, it is not necessarily necessary to use a container with low self-fluorescence for fluorescence immunoassay, but if it is possible to efficiently collect more of the fluorescence emitted by the sample, it is possible to reduce the weak fluorescence. However, there are many potential needs for this, as even more sensitive measurements can be made.

In order to meet these needs, the present inventors investigated various methods and discovered that measurement sensitivity could be increased by providing a layer that reflects fluorescent light toward the inside of the container, and conducted extensive research. This led to the completion of the present invention.

[Purpose of the invention]

That is, an object of the present invention is to provide a container for fluorescence immunoassay that enables highly sensitive measurements.

[Structure of the invention]

The present invention is a container for holding a fluorescent sample, comprising:
This is a container for fluorescence immunoassay, characterized in that a layer that reflects fluorescent light is formed on the outer surface or inner layer that does not come into contact with the sample.

Container shapes include microplate, Kievet,
There are test tubes, spitz, reaction plates, etc., but they are selected depending on the purpose and are not particularly limited.

In fluorescence immunoassay, proteins such as the above-mentioned antigens and antibodies are adsorbed and immobilized on the surface of a container and a reaction is carried out, but in general, the adsorption of proteins to metals is unstable and uniform and reproducible results are not possible. I can't hope. On the other hand, adsorption to plastic is stable, and if a container with a plastic surface is used, highly accurate measurements can be made. Therefore, the surface on which proteins are adsorbed and the reflective layer must be separate layers. For example, a container that achieves these conditions may include a reflective rfI on the outer surface of the plastic molded product (1) shown in Figure 1.
Container provided with I (2), molded container (4) shown in Figure 2
A container (i.e., a container with a reflective layer (2) on its inner surface and a plastic surface layer (3) on top of the reflective layer (2))
) can be used.

The layer (2) that reflects fluorescent light is made of metal or a substance containing metal.

Metals that can be used in the present invention include silver, mercury,
In addition to gold, platinum, copper, aluminum, nickel, soot, titanium, chromium, indium, etc., mercury amalgam and alloys of the above metals may also be used. Examples include silver R-st dispersed in a metal paint, metal paint, and the like. A method for forming a reflective layer (2) of metal or a substance containing metal on the surface of a plastic molded product (1) or a molded container (4) is as follows:
Methods include metal plating, vapor deposition, snootering, ion blating, and coating with a substance containing metal, but are not limited to these methods.

Methods for forming the plastic surface layer (3) on the surface of the reflective M (2) include coating a plastic solution, emulsion, etc., or dipping the container therein, as well as forming a siplastic layer by plasma polymerization, etc. A method of coating with can also be used.

Plastic molded product (1) and plastic surface layer (
The plastic forming 3) may be any transparent material with low self-fluorescence, but preferred examples include polystyrene, vinyl chloride, and polymethyl methacrylate. There is no problem even if the surface is treated to improve adsorption of proteins such as antigens and antibodies. Further, in the present invention, since fluorescent light is reflected and amplified through the plastic layer of the container, it is preferable that the layer be thin in order to reduce loss of fluorescent light within this layer.

Further, the thickness of the metal layer serving as the reflective layer (2) is not particularly limited as long as it maintains a reflective function. The material for forming the molded container (4) is not particularly limited, and may include glass, ceramic, metal, etc. in addition to plastic. Even when plastic is used, the above-mentioned restrictions on self-fluorescence and transparency do not apply, and various plastics including ethylene, polypropylene, etc. can be used. Further, when metal is used, if the inner surface is finished to a mirror finish, there is an advantage that the formation of the reflective layer (2) becomes unnecessary. Moreover, when the reflective layer (2) in FIG. 2 is formed using silver yeast, metal yeast, etc., its surface is substantially made of a resin layer (plastic), and the plastic surface N (3) It is possible to omit the formation of .

〔Effect of the invention〕

By using the container for fluorescence immunoassay of the present invention, highly sensitive measurements are possible. That is, the reflective layer according to the present invention reflects the fluorescent light emitted by the measurement sample, thereby increasing the fluorescent light intensity perceived by the measuring instrument. This makes it possible to measure with high precision even in the extremely small amount range, which was conventionally too weak to measure accurately.

The present invention will be explained in more detail below with reference to comparative examples and examples.

Reference Example 1> Self-fluorescence was compared using microplates made of pyristyrene and pyrovinyl chloride from various companies as containers for fluorescence immunoassay.

The fluorescence measurement device was a Microfluor (manufactured by Dynatic).
Micro FLUOR (trade name) was used. The excitation wavelength and fluorescence wavelength were 365 nm and 450 nm, respectively. This wavelength corresponds to the measurement wavelength of the typical fluorescent reagent 4-methylumbelliferone.

The results are shown in Table 1, and it can be seen that most of them, with a few exceptions, have low self-fluorescence.

Table 1 Microplate made of pyristyrene Microplate made of polyvinyl chloride <Example 1> As a plastic container, a plate aminated type for Eliza made by Sumitomo Bakelite@ (made by Zerystyrene, product number MS-
3696F) to form a fluorescent reflective layer thereon.

As a reflective layer, a thin aluminum film was formed on the outer surface of the molded product by treating it at 5 mA for 120 minutes using a Nippon Shinku Kokuri Co., Ltd. snow zeta ringing device.

To each well of the plate, 100 µ of an aqueous solution of 4-methylumbelliferin-6-elon at a concentration of 10-10 M was added. Using the same fluorescence measuring device as in Reference Example 1, the fluorescence intensity was measured at the same wavelength. As a comparative example, similar measurements were conducted using a plate on which no reflective layer was formed. Second
Table 2 <Example 2> The outer surface of the molded product was plated with silver using a microplate manufactured by Flow Co., Ltd. (made of irivinyl chloride, kite type) as shown in the table.

Add β-D-galactosidase (manufactured by Sigma, hereinafter referred to as β-Gat) to this plate.
The solution was placed at a concentration of 100 liters/well and left at 4°C for 18 hours. After washing with saline, add 3X10
M 4-methylumbelliferyl-β-D-galactoside (manufactured by Kotzholite) was added 50) d/well and allowed to react at room temperature for 30 minutes. The reaction was stopped by adding 2'00 μt/well of glycine-sodium hydroxide buffer (PH 10.3), and measurements were carried out in the same manner as in Example 1. As a comparative example, similar measurements were performed on a plate that was not subjected to plating treatment. As shown in Table 3, it can be seen that the reflective layer of the present invention has a great effect.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 show an embodiment of the present invention, and FIG. 2 shows the space between the molded container and its inner plastic surface layer (inner layer)
This is a container with a reflective layer on the inside.

Claims (3)

[Claims] (1) A container for fluorescence immunoassay, which is a container for holding a sample that emits fluorescence, and is characterized in that a layer that reflects fluorescence is formed on the outer surface or inner layer that does not come into contact with the sample. (2) The container for fluorescence immunoassay according to claim 1, wherein the layer that reflects fluorescent light is made of a metal or a substance containing metal. (3) The container for fluorescence immunoassay according to claim 1, characterized in that the surface in contact with the sample is made of plastic.