JPH0628591B2 - Dot blot method and reactor therefor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a dot blot method for detecting DNA, RNA, protein, etc. in a sample and a reactor therefor.

[Prior Art] Conventionally, the dot blot method has been widely used to detect an antigen or an antibody (hereinafter referred to as a test substance) such as DNA, RNA, or protein in a sample.

The dot blot method includes a method using a radioisotope as a label and a method using an enzyme. The method using a radiolabel has the advantage of high detection sensitivity, but requires large-scale special equipment for handling radioactive substances, and poses problems such as exposure hazard and disposal of radioactive waste.

On the other hand, the method using the enzyme label has an advantage that the above-mentioned various problems caused by the radioactive substance are not used because no radioactive substance is used. The conventional dot blot method using an enzyme is performed as follows. First, a sample containing a test substance is spotted on a filter made of nitrocellulose, nylon, or the like. This is usually carried out by setting a filter in the dot blot apparatus, putting the sample in the hole of the dot blot apparatus, and sucking the sample to adsorb the test substance in the sample onto the filter. After fixing the test substance to the filter by a method such as baking the filter,
The filter is subjected to blocking treatment to prevent non-specific adsorption, and after washing, the test substance is reacted with the probe and then washed. When the test substance is present on the filter, the test substance and the probe are specifically bound by hybridization or an antigen-antibody reaction. When the test substance is not present on the filter (that is, when the sample does not contain the test substance, the probe does not specifically bind to the filter and is removed from the filter by washing. In some cases, a low molecular weight biotin is bound to the probe in order to further enhance the reaction sensitivity, and the avidin-labeled enzyme conjugate is allowed to act after the reaction between the test substance and the probe. In any case, the method of washing is followed.In any case, when the test substance is present on the filter, the labeled enzyme is indirectly bound to the filter, and when the test substance is not present on the filter, the labeled enzyme is filtered. Therefore, the presence of the test substance in the sample can be detected by detecting the labeled enzyme on the filter, or the It can be carried out determination of analyte by measuring.

As the labeling enzyme used in the conventional dot plotting method, those that directly or indirectly generate a dye by an enzymatic reaction, such as peroxidase, β-D-galactosidase, and alkaline phosphatase, are usually used.
The detection or quantification of the labeling enzyme is carried out by detecting or quantifying the produced dye. The substrate solution for the enzymatic reaction is applied to the filter.

The dot blot method using the above-mentioned conventional enzyme label has an advantage that the above-mentioned various problems in the method using the radiolabel do not occur because the radioactive substance is not used, but on the other hand, the measurement is more effective than the method using the radiolabel. There is a problem of low sensitivity. In addition, the dot blot method is usually
A large number of spots (96, for example) are provided on one filter, and the spots on one filter are processed at the same time. However, when the substrate solution is applied, there is an interaction between the spots and the measurement sensitivity is high. There was also a problem that it would decrease.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a dot blot method using an enzyme label having a higher measurement sensitivity than the conventional method.

[Means for Solving the Problems] As a result of earnest research, the inventors of the present application have performed a step of causing a substrate solution to act on a labeled enzyme by using a special reactor, so that the measurement sensitivity is higher than that of the conventional method. The inventors have found that it can be increased and completed the present invention.

That is, the present invention, in the dot blotting method using an enzyme label, the step of causing the substrate solution to act on the labeled enzyme spotted on the filter is prevented from leaking to the bottom plate, the through hole and the bottom surface of the peripheral portion of the through hole. And a body plate having a thickness capable of retaining a liquid in the through hole, and the filter so that the spot of the labeled enzyme is surrounded by the liquid leakage prevention member of the through hole. There is provided a dot blot method, which is characterized in that the through hole is filled with a substrate solution to carry out an enzymatic reaction.

Furthermore, the present invention provides a bottom plate, a through-hole, and a main body plate that is detachably laminated on the bottom plate and has a thickness capable of holding a liquid in the through-hole; Provided is a reactor for dot blot having a liquid leakage prevention member provided on the bottom surface of the peripheral portion of a through hole.

[Detailed Description of the Invention] The method of the present invention can be applied to any dot blotting method using an enzyme label, and therefore, as a test substance, a substance having antigenicity such as DNA, RNA and protein and polysaccharides can be used. And antibodies.

The characteristic part of the present invention lies in the step of reacting the labeling enzyme retained on the filter with the substrate solution, and the steps before that are not limited at all, and any method can be adopted. Further, the enzyme used for labeling is not particularly limited, and any conventionally used enzyme can be adopted. A particularly efficient method is a method in which a sample is adsorbed using a commercially available 96-well dot blot apparatus (96 spots are formed on one filter by this method).

In the method of the present invention, the labeled enzyme retained on the filter is measured by using a specific reactor as described in the section of the prior art. An example of this reactor is shown in FIG.
In the example shown in FIG. 1, the reactor 10 has a bottom plate 12 and a body plate 14 laminated on the bottom plate 12. The body plate 14 is provided with a through hole 16. The body plate 1 has a thickness capable of holding the liquid in the through hole 16. The number of through holes is the same as the number of spots on one filter, and the position where the through holes 16 are provided is also 1
It corresponds to the position of the spot on the filter. Therefore, for example, when the spots are formed by using a commercially available 96-well dot blot apparatus, the number and size of through holes corresponding to the holes of the 96-well dot blot apparatus are the same as those of the holes of the dot blot apparatus. It is provided at a position corresponding to the position. A liquid leakage prevention member 18 is provided on the bottom surface of the peripheral portion of the through hole 16. The liquid leakage prevention member 18 may be, for example, an O-ring, or a convex edge integrally formed with the body plate 14. The reactor 10 preferably further has means (not shown) for fixing the body plate 14 and the bottom plate 12 together. Such a fixing means may be, for example, a screw hole and a screw penetrating the main body plate and the bottom plate, or one end of the main body plate and the bottom plate may be openably and closably attached by a hinge or the like, and the other end may be sandwiched by a clip or the like. It may be fixed by or fixed by screw.

The enzymatic reaction is performed as follows. First, the bottom plate 1 is provided with the filter 20 in which the labeling enzyme is held at the spot position.
It is sandwiched between 2 and the body plate 14 and fixed. In this case, each spot 22 on the filter corresponds to the through hole 16
The liquid leakage prevention member 18 provided on the bottom surface of the peripheral edge portion of the. This can be easily done by making the position and size of the through hole 16 provided in the main body plate 14 coincident with the position and size of the hole of the dot blot apparatus when forming a spot using the dot blot apparatus. Can be achieved. In the above state, the filter 20 is placed between the main plate 14 and the bottom plate 16.
After fixing, the through hole 16 is filled with the substrate solution. Then, an enzyme reaction occurs with the labeling enzyme retained on the spot, and a signal such as a dye is generated according to the type of the enzyme.Therefore, the amount of the labeling enzyme, that is, the amount of the labeling enzyme in the sample by conventional colorimetric measurement, etc. The amount of test substance can be measured.
If the through holes of the main body plate are provided at the same position and size as the commercially available microplate, 96 commercially available luminometers can be used to automatically measure 96 spots in an extremely short time, so it is efficient. The measurement can be performed (note that the commercially available dot blot apparatus has holes of the same size and the same position as those of the commercially available microplate).

[Effects of the Invention] In the method of the present invention, the substrate solution is filled in the holes of the main body plate to carry out the enzymatic reaction, so that a large amount of the substrate is reacted as compared with the case where the filter is applied as in the conventional method. be able to. Therefore, the amount of dye or the like produced by the enzymatic reaction is increased, and the sensitivity of measurement is improved. Further, in the method of the present invention, the interaction between the spots is prevented by the action of the liquid leakage prevention member at the stage of acting the substrate solution, and the measurement sensitivity based on the interaction between the spots does not decrease. Also, as the main body plate of the reactor of the present invention. By using a microplate that has been used for immunoassays, etc., excluding the bottom plate, by using a commercially available luminometer,
The measurement can be automated. Furthermore, in the assay using a microplate, which has been widely performed in the past, an error between wells due to non-specific adsorption may not be negligible, but according to the method of the present invention, one filter unit is used. Since the processing is performed, such an error does not occur.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional view of one embodiment of the reactor of the present invention used in the dot blot method of the present invention. 10 ... Reactor, 12 ... Bottom plate, 14 ... Main body plate, 16 ... Through hole, 20 ... Filter, 22 ... Spot

Claims (3)

[Claims] 1. In a dot blotting method using an enzyme label, a step of causing a substrate solution to act on a labeled enzyme spotted on a filter is performed by a liquid leakage prevention member on a bottom plate, a through hole and a bottom surface of a peripheral portion of the through hole. And a body plate having a thickness capable of holding a liquid in the through hole,
The dot blotting method, wherein the filter is sandwiched so that the spot of the labeled enzyme is surrounded by the liquid leakage prevention member in the through hole, and the through hole is filled with a substrate solution to carry out an enzyme reaction. . 2. A bottom plate, a body plate provided with a through hole and having a thickness capable of holding a liquid in the through hole, the body plate being removably laminated on the bottom plate, and the through hole of the body plate. And a liquid leakage prevention member provided on the bottom surface of the peripheral portion of the dot plot reactor. 3. The reactor according to claim 2, further comprising means for fixing the bottom plate and the body plate.