JP4579752B2 - Specimen spotting method using spotting support - Google Patents

Description

The present invention belongs to a technical field related to a spotting method in which a sample is easily added.

Conventionally, a silver-stained high-sensitivity trace protein quantification method using a cellulose acetate membrane has been used as a method for quantitatively quantifying a small amount of protein with high sensitivity and ease (Patent Document 1). This is a certain amount of urine on the porous polymer membrane in the dry state, spotted analytes cerebral spinal fluid, etc., and the visible and specifically coupled to form the silver on immobilized trace protein, densitometry This is a method of measuring the concentration with a tomometer . In addition, in order to obtain an accurate measurement result using a densitometer, it is known that a certain amount of sample needs to be spotted on a certain area of the cellulose acetate film without being biased or distorted (Non-Patent Document 1). , P104-105).

For spotting, the tip of the tip 12 or the capillary glass tube 4 attached to the micropipette is immersed in the specimen 6 and sucked (FIG. 2a) , and the tip of the tip 12 or the cut end of the tube 4 is brought into contact with the support 1 vertically as it is ( FIG. 2b) is carried out by absorbing the specimen on the support 1 (FIGS. 2c and d) .

To place a certain amount of sample on a certain area without deviation or distortion, that is, to make the image after spotting always a uniform circle with the same size, weigh exactly 3 microliters, The end of the micropipette or capillary glass tube may be kept in vertical contact with a constant force until all of the specimen sucked into the capillary glass tube is absorbed by the support (FIGS. 2b and 2c) . At this time, if the support is damaged, the density of the portion changes. Therefore, the contact force must be adjusted so as not to damage the support.
Japanese Patent Laid-Open No. 2002-236127 Hisiko Hashimoto “Cellulose Acetate Membrane Electrophoresis”, Clinical Examination Vol. 26, No. 1, p99-107, Medical School, 1982

Repeating spotting a large number of times, especially holding a capillary glass tube that is easy to break, is very complicated.

In addition, it is necessary to be skilled to make the image after spotting always have a uniform circular shape with no unevenness and to adjust the force so as not to damage the support.

The present invention provides a method for spotting a predetermined amount of a sample in a circular shape by applying a process in which the sample does not soak in other than the sample absorbing portion of the support.

The processing is performed by drawing an annular shape in which the specimen does not permeate around the portion where the specimen is to be absorbed by rotating the cylinder with an inner diameter of 7 millimeters and the outer diameter being pressed against the cellulose acetate film while rotating vertically. Alternatively, the seal part width of the film sealer may be formed in a circular shape having an inner diameter of 7 mm and brought into contact with the cellulose acetate membrane. Furthermore, processing that does not allow water to permeate may be applied to all parts except the perfect circle part that absorbs the specimen.

By simply placing a certain amount of specimen at any location inside the ring where the specimen drawn by processing does not permeate, the image after spotting can be made into a perfect circle of the same size with no unevenness. I was able to spot it reliably. In addition, since it is no longer necessary to bring the pipette or capillary glass tube into contact with the support, unevenness in which the concentration increases only at the contact portion can be prevented.

[Example 1]
The inner diameter is 7 millimeters, and the outer diameter is pressed against the cellulose acetate membrane 1 while rotating an arbitrary cylinder 8 vertically (FIG. 3a) , and the periphery of the portion to absorb the specimen is denatured by pressure (FIG. 3b). A wearing support was prepared. As an example, a spotting support for 48 specimens is shown in FIG. Next, 3 microliters was accurately weighed with a micropipette to which the chip 12 was attached, and dropped onto the specimen absorption part without contacting the tip- attaching support on which the tip of the chip 12 was produced (FIG. 5a) . Regardless of the location where the specimen is dropped is the central portion of the specimen absorbing portion or a portion deviated from the center (FIG. 5a) , the specimen spreads and is absorbed as a perfect circle of the same size (see FIG. 5a) . 5b) .

[Example 2]
The sealer width of the film sealer is formed into a circular shape with an inner diameter of 7 mm to form a sealer 10, which is energized and brought into contact with the cellulose acetate membrane (FIG. 4 a) to absorb the specimen of the membrane. The sample was heat-denatured in an annular shape so that the sample did not penetrate (FIG. 4b). Next, in the same manner as in Example 1, 3 microliters was accurately weighed with a micropipette equipped with a chip 12, and dropped onto the specimen absorption part without contacting the tip support that produced the tip of the chip 12 (see FIG. 5a) As a result, the specimens spread and absorbed as a perfect circle of the same size.

Silver-stained high-sensitivity trace protein quantification method can be easily performed, and subsequent pathological analysis by protein fractionation can be performed more easily, contributing to diagnostic treatment and research of renal diseases.

It is a top view of a spot wearing support. It is explanatory drawing of the conventional spotting method. 3 is an explanatory diagram of a spotting support manufacturing method of Example 1. FIG. It is explanatory drawing of verification of the effect of Example 1. FIG. FIG. 5 is an explanatory diagram of a spotting support manufacturing method of Example 2.

DESCRIPTION OF SYMBOLS 1 Support body 2 The ring drawn in by the sample drawn by processing 3 Sample absorption part
4 Capillary glass tube
5 test tubes
6 specimens
7 Sample absorbed and supported by the support
8 cylinder
9 Parts denatured by pressure
10 Sealer
11 Heat-denaturing part
12 chips

Claims (1)

In a method in which a fixed amount of urine or cerebral spinal fluid corresponding to the area is spotted on a fixed area of a porous polymer membrane in a dry state, immobilized, visible, and the concentration is measured. The membrane is pre-denatured so that the specimen does not soak in other than the specimen-absorbing portion. The membrane is subjected to processing as a single membrane, leaving a round-shaped specimen-absorbing portion and denatured by heat or pressure. It is characterized by using a spotting support formed by performing a certain process, and characterized in that a certain amount of specimen is added without the specimen spotting tool contacting the spotting support , A specimen spotting method that makes it possible to absorb specimens without bias, distortion, and unevenness in a circular shape with a constant area.

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