JP2023122235A - Specimen pretreatment reagent in freeze-dried state and measuring method - Google Patents

Abstract

To provide a specimen pretreatment reagent in a freeze-dried state without needing any complicated measurement operation, with excellent measurement reproducibility, and used for extracorporeal diagnostic agent and the like.SOLUTION: A specimen pretreatment reagent in a freeze-dried state freezes an aqueous solution containing an alkali agent or a reducing agent and an excipient after dispensing the aqueous solution in a container, further dispenses an aqueous solution containing an agent different from the previously added agent and an excipient among the reducing agent and alkali agent in the container while keeping a frozen state, and freezes and dries the aqueous solution.SELECTED DRAWING: Figure 1

Description

The present invention provides a freeze-dried specimen pretreatment reagent used for liberating vitamins in a freeze-dried state for use in in vitro diagnostics and the like, wherein the freeze-dried reagent containing an alkaline agent and a reducing agent is used for two-step dispensing. The present invention relates to improving the reproducibility of measurement without requiring complicated measurement operations by fabricating the measurement device in a single step.

Specimen pretreatment reagents used for in vitro diagnostics and the like are widely used in fields such as clinical examinations as specimen pretreatment liquids in liquid form. This sample pretreatment liquid, which is in liquid form, can be used as it is, so that the burden on the operator is light, but it is disadvantageous in terms of transportation due to its volume and weight. Also, since they are usually supplied and used as bulk products, they are susceptible to environmental temperature and humidity. In other words, since it is used in an unsealed state, the effect of concentration cannot be avoided, and the concentration of the active ingredient in the sample pretreatment solution fluctuates with the duration of use, and the accurate measurement value fluctuates with the duration of use. It is feared that it will continue.

Vitamins also include small molecules such as vitamin D metabolites, vitamin B12 and folic acid. Since most of these small molecules in blood are bound specifically or non-specifically to proteins, lipids, etc., to measure these small molecules, the small molecules must be released from the proteins, etc. There is a need. Each vitamin has a specific binding protein, which contributes to its stability in blood. For example, vitamin D binding protein (DBP), haptocorrin and intrinsic factor are well known.

Alkaline treatment and reduction treatment have been reported as conventionally known methods for releasing vitamins from binding proteins and the like, and sodium hydroxide aqueous solution is often used as an alkaline agent. Dithiothreitol (DTT) is often used as a reducing agent to stabilize denatured binding proteins. However, since the reducing power of the reducing agent decreases in an aqueous solution, especially in an alkaline aqueous solution, it is freeze-dried and stored in a separate container from the alkaline agent. Therefore, before releasing the vitamins from the binding proteins, etc., it is common to dissolve either or both of the reducing agent and the alkaline agent in water, and then mix them before using them for the release. . However, such a conventional method is complicated, and has problems of lowering the reproducibility of release of vitamins from binding proteins and the like and deteriorating the accuracy of vitamins measurement. Therefore, methods have been proposed to solve these problems.

Patent Document 1 reports a freeze-dried specimen pretreatment reagent for use in an automatic analyzer. However, Patent Document 1 does not describe the composition or manufacturing method of the sample pretreatment reagent. Sugars, proteins, and the like are used as excipients in various reagents in lyophilized form. When sugars and proteins are low, it is difficult to maintain the shape. Fluctuations and deviations from accurate measurements are a problem. If there are many sugars or proteins, it is possible to maintain the shape, but if the ingredients and concentration are not appropriate, the solubility after re-dissolution will be poor, resulting in poor measurement reproducibility. It has been a problem that it hinders highly accurate measurement of the target component.

Japanese Patent No. 5811244

In the field of clinical testing, various reagents are required to have reproducibility during reagent measurement. SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a freeze-dried specimen pretreatment reagent for use as an in-vitro diagnostic agent or the like, which does not require complicated operations and has good measurement reproducibility.

As a result of intensive studies to solve the above problems, the present inventors have found that by optimizing the method for producing a sample pretreatment reagent in a freeze-dried state, no complicated operation is required and the measurement reproducibility is good. It found out that it becomes and came to complete this invention.

That is, the present invention is as follows.
(1) dispensing an aqueous solution containing an alkaline agent and an excipient into a container and then freezing;
A sample pretreatment reagent in a freeze-dried state, wherein an aqueous solution containing a reducing agent and an excipient is further dispensed into the container while maintaining the frozen state, and then freeze-dried.
(2) dispensing an aqueous solution containing a reducing agent and an excipient into a container and then freezing;
A sample pretreatment reagent in a freeze-dried state, characterized by further dispensing an aqueous solution containing an alkaline agent and an excipient into the container while maintaining the frozen state, and then freeze-drying.
(3) The specimen pretreatment reagent according to (1) or (2), wherein the excipient is a saccharide.
(4) The sample pretreatment reagent according to any one of (1) to (3), wherein the saccharide is selected from sucrose and mannitol.
(5) A freeze-dried reagent stored in a container having two storage holes in the same container,
In one hole, the specimen pretreatment reagent according to any one of (1) to (4),
A specimen pretreatment reagent in which a neutralizer is stored in a freeze-dried state in the other hole.
(6) A method for measuring vitamins using the specimen pretreatment reagent according to any one of (1) to (5).

The present invention will be described in detail below.

The sample pretreatment reagent of the present invention is used to release small molecules to be measured from proteins, lipids, and the like. Before use, add a dissolution solution to dissolve the solution and use it as a sample pretreatment solution.

The subject to be measured using the specimen pretreatment reagent of the present invention is not particularly limited, but examples thereof include 25-hydroxyvitamin D, vitamin B12, folic acid, and the like.

The sample pretreatment reagent in a freeze-dried state according to the present invention is obtained by dispensing an aqueous solution containing a first component and an excipient into a container and then freezing the solution. and excipients (also referred to as two-step dispensing), followed by freeze-drying. By this manufacturing method, a freeze-dried cake containing the first component on the bottom side and a frozen cake containing the second component on the top side are formed without mixing the first component and the second component in a liquid state. A dry cake is formed, and deterioration of the reducing agent due to the alkaline agent can be prevented. An alkaline agent may be selected as the first component and a reducing agent as the second component, or vice versa, i.e. selecting a reducing agent as the first component and an alkaline agent as the second component. You may choose.

In the present invention, the alkaline agent is preferably a strongly alkaline agent such as sodium hydroxide or potassium hydroxide. Only one type of alkaline agent may be used, or two or more types may be used in combination.

In the present invention, any reducing agent may be used as long as it stabilizes a sample denatured by an alkaline agent, such as dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine Hydrochloride (TCEP-HCl). Only one type of reducing agent may be used, or two or more types may be used in combination.

In the present invention, saccharides and proteins are preferred as excipients, and saccharides are particularly preferred. As sugars, sucrose, mannitol, xylitol, sorbitol, galactitol, ribitol and the like are preferable, and sucrose and mannitol are particularly preferable. Only one type of saccharide may be used, or two or more types may be used in combination.

The freeze-dried specimen pretreatment reagent of the present invention may contain a constituent of a buffer solution. Further, surfactants and salts may be contained. Although they are not particularly limited, anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants can be used as surfactants. Examples of constituents of the buffer solution include constituents such as Tris, MOPSO, MOPS, MES, phosphoric acid, and carbonic acid. Salts include, for example, sodium chloride, potassium chloride, magnesium chloride, and zinc chloride. can do. In addition to these, the freeze-dried sample pretreatment reagent can coexist with other reagent components, etc., if necessary.

When measuring small molecules such as vitamins according to the present invention, it is preferable to add a neutralizing agent to initiate an immune reaction in order to prepare the measurement environment. Neutralizing agents include phosphoric acid and citric acid. It is more preferable to provide this neutralizing agent in a freeze-dried form, not in a liquid state, from the viewpoint of preventing deterioration of reagent performance due to concentration or deterioration of the neutralizing agent. The type and concentration of the neutralizing agent can be appropriately determined in consideration of the concentration of the alkaline agent and the optimum pH for immune reaction.

Furthermore, when the container is a container having a plurality of holes, the freeze-dried reagent prepared by two-step dispensing of the alkaline agent and the reducing agent of the present invention is filled in one hole, and the neutralizing agent is filled in the other hole. Can be provided as lyophilized. Although the number of holes in the container is not limited, if a two-hole container is used, one hole contains the freeze-dried alkali agent and reducing agent, and the other hole contains the freeze-dried neutralizer. It is more preferable for the miniaturization of the device and the operation in an automatic analyzer.

In the freeze-dried sample pretreatment reagent of the present invention, the freeze-dried reagent containing an alkali agent and a reducing agent is prepared by two-stage dispensing, thereby suppressing the deterioration of the reducing agent due to the alkali agent and making it complicated. It does not require any special measurement operations. Therefore, if the sample pretreatment reagent in a freeze-dried state of the present invention is used as an in-vitro diagnostic agent or the like, measurement can be performed with high accuracy.

FIG. 4 is a diagram showing the configuration of a freeze-dried reagent using the two-hole container of the example.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In addition, using a fully automatic enzyme immunoassay device AIA-CL2400 manufactured by Tosoh as an immunoassay device, using CL AIA-PACK 25-OH Vitamin D for the device as an immunoassay reagent, each measurement by a one-step delay competitive method. did Each specimen pretreatment reagent was prepared as described later.

(Examples 1 to 5, Comparative Example 1)
Various solutions were prepared as shown in Table 1. Details are as follows.

Sodium hydroxide was added to pure water to give a concentration of 0.3 mol/L. An additional 5% (weight/volume) scroll was added and dispensed into one hole of a two-hole reagent container and frozen at -70°C for 1 hour. This reagent container has two identical holes. Furthermore, a solution containing saccharides and a reducing agent TCEP-HCl having the composition shown in Table 1 was prepared and dispensed into the same hole as the sodium hydroxide solution was dispensed to the tip of the reagent container ( Two-stage aliquot) and frozen at -70°C for an additional hour.

Next, potassium dihydrogen phosphate was added to the pure water to give a concentration of 0.2 mol/L. Furthermore, 10% (weight/volume) of mannitol and 0.01% (weight/volume) of Tween 20 were added and dispensed into the holes different from the above-mentioned alkaline agent and reducing agent. After freezing for another hour. Lyophilization was performed.

In Comparative Example 1, only the alkaline agent was used, and freeze-drying was performed without dispensing the reducing agent.

(Measurement reproducibility test)
Using human serum with a 25-hydroxyvitamin D concentration of 30 ng/mL as a sample, a solution (dispensed water containing sodium azide) was added to the freeze-dried sample pretreatment reagent prepared in Examples 1 to 5 and Comparative Example 1. was added and dissolved to prepare a sample pretreatment solution. Specimens were pretreated with it and 25-hydroxyvitamin D concentrations were measured. A series of these operations were performed by the automatic immunoassay device. The sample pretreatment operation was performed by automatic pretreatment on the apparatus. A series of operations for measuring the 25-hydroxyvitamin D concentration after pretreatment was repeated 45 times, and the mean value of the 25-hydroxyvitamin D measurements was obtained. Further, based on the measured values, the measurement reproducibility was calculated. Table 2 shows the results.

As is clear from Table 2, in Comparative Example 1, the reproducibility was poor with a CV of 8.6%. This denatures the binding protein in the blood with sodium hydroxide and releases 25-hydroxyvitamin D, but since no reducing agent is added, the denatured binding protein cannot be stabilized, and the measurement result is This is thought to be due to the influence of

On the other hand, in Examples 1 to 5, CV 1.6% to 2.7%, showing good measurement reproducibility regardless of the concentration of the saccharide added in the second stage. This was because the reducing agent was lyophilized in two steps, which prevented the alkaline agent and reducing agent from mixing until just before the measurement, and the performance of the reducing agent did not deteriorate. It is considered that this is because the stability could be achieved.

1: Sample pretreatment reagent supply containers 2a, 2b: Supply container opening 3a: Alkaline agent 3b: Reducing agent 3c: Neutralizing agent 4: Aluminum seal

Claims (6)

After dispensing an aqueous solution containing an alkaline agent and an excipient into a container, freezing it,
A sample pretreatment reagent in a freeze-dried state, wherein an aqueous solution containing a reducing agent and an excipient is further dispensed into the container while maintaining the frozen state, and then freeze-dried. After dispensing an aqueous solution containing a reducing agent and an excipient into a container and freezing it,
A specimen pretreatment reagent in a freeze-dried state, characterized by further dispensing an aqueous solution containing an alkaline agent and an excipient into the container while maintaining the frozen state, followed by freeze-drying. 3. The specimen pretreatment reagent according to claim 1, wherein said excipient is a saccharide. The specimen pretreatment reagent according to any one of claims 1 to 3, wherein said saccharide is selected from sucrose and mannitol. A freeze-dried reagent stored in a container having two storage holes in the same container,
In one hole, the sample pretreatment reagent according to any one of claims 1 to 4,
A specimen pretreatment reagent in which a neutralizer is stored in a freeze-dried state in the other hole. A method for measuring vitamins using the specimen pretreatment reagent according to any one of claims 1 to 5.