JPH05172816A - Measuring method for antigen - Google Patents

Abstract

PURPOSE:To enable the highly accurate measurement of an antigen in a short time by applying force to an obtained sediment in another direction different from the sedimentation direction upon applying centrifugal force, followed by measuring its dimensional change. CONSTITUTION:A specimen, in which the antigen to be measured is likely to be contained, is reacted with a carrier, in which the corresponding antibody is contained in a solid phase, directly or dilutedly. An antigen-antibody reaction can be performed under a normal condition. The resulted reactant is forcibly sedimented by a centrifugal operation to form a sediment of a reactant. The condition for the centrifugal operation is not preferably restricted as far as the reactant can be sedimented, for example about ten minutes at 1200 rpm. Next, an external force is applied to the obtained sediment in another direction different from the sedimentation direction upon applying the centrifugal force, followed by measuring the dimensional change of the sediment. Although the sediment maintains the pellet-like shape if an antigen-antibody reaction has taken place, it can be observed that the carrier particle flows in a tear-like fashion if no antigen-antibody reaction has taken place.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immunologically measuring an antigen by an agglutination reaction using a carrier.

[0002]

2. Description of the Related Art In recent years, clinical laboratory diagnostic methods using immunological methods have been frequently used because of their specificity and high sensitivity. Among them, the agglutination reaction using a carrier on which an antibody is solid-phased has been conventionally used because it is simple and highly sensitive. As a method that has been conventionally used, a reverse passive hemagglutination reaction (RP
HA method). Similarly, a method of using a synthetic carrier such as latex particles or gelatin particles as a carrier is also widely used as a method of measuring an antigen or an antibody.
In all of these conventional methods, the antigen-antibody reaction was determined by allowing the reaction product to stand and spontaneously precipitating the agglutination image to measure the antigen.

However, in the conventional measurement based on the determination of the agglomeration image of spontaneous sedimentation, it took several hours to make the determination, and it was not possible to meet the demand for obtaining test results quickly. Then, for example, a method of shortening the sedimentation time by using a carrier having a large specific gravity and a method of shortening the sedimentation time by magnetic force by using carrier particles containing a magnetic material have been devised. However, these methods are insufficient in the effect of shortening the time, and there is a problem that the agglutination image becomes unclear due to the change of the carrier and the non-specific reaction increases, and sufficient results have not been obtained. .

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method capable of highly sensitively measuring an antigen in a shorter time than conventional methods.

[Means for Solving the Problems]

The present inventors have studied the agglutination reaction for the purpose of improving the disadvantage that the reaction time in the conventional antigen measurement method by agglutination reaction is long and developing a highly sensitive assay method. , Reacting the carrier particles on which the antibody corresponding to the antigen to be measured is solid-phased with the sample, forcibly collecting the reactants by centrifugation to form a precipitate, and then sedimenting it if an operation such as tilting is added The inventors have found that the method for measuring the change in the shape of an object is significantly superior to the methods conventionally found, and completed the present invention.

That is, in the present invention, in the method for measuring an antigen using an agglutination reaction, carrier particles having an antibody corresponding to the antigen to be measured solid-phased are reacted with a sample, and the reaction product is forcibly centrifuged. Provided is a method for measuring an antigen, which comprises subjecting the obtained sediment to a force in a direction different from a sedimentation direction at the time of centrifugation to measure the shape change thereof.

In the present invention, carrier particles on which an antibody corresponding to the antigen to be measured is solid-phased are used. As the carrier, animal red blood cells such as sheep and chicken, which are usually used for agglutination reaction, and polysaccharide polymer. , Latex particles, gelatin particles and the like can be used. In addition, the solid phase method of the antibody may be any conventionally used method such as physical adsorption, chemical bonding, or embedding on the surface of a carrier.

As a measuring method, a sample which may contain an antigen to be measured is directly or diluted and the corresponding antibody is reacted with a solid phase carrier. The antigen-antibody reaction can be performed under normal conditions. The reaction product is forcibly settled by a centrifugal operation to form a precipitate of the reaction product. The condition of centrifugation is not particularly limited as long as it can precipitate the reaction product, and is, for example, 1200 rpm for about 10 minutes.

Next, an external force is applied to the obtained sediment in a direction different from the sedimentation direction at the time of centrifugation, and the shape change of the sediment is measured. The simplest and preferred method of applying force is
The settling is performed by inclining or suspending the settling at an angle of 30 to 90 degrees, preferably 75 degrees with respect to the settling direction and allowing the settling to stand. The standing time is not particularly limited, but is, for example, about 5 minutes. Further, when magnetic particles are used as the carrier, a force can be applied to the sediment by applying the ground in the above direction.

If an antigen-antibody reaction has occurred, the precipitate remains in the form of pellets, but if no antigen-antibody reaction has occurred, it can be observed that the carrier particles flow in a tear shape. That is, the strength of aggregation due to the antigen-antibody reaction is judged by the susceptibility of the reactants, which have been forcibly collected by centrifugation in advance, to collapse. This determination can be sufficiently made even by visual observation, and can also be made by an image device.

[0011]

According to the method of the present invention, the time required for the measurement is drastically shortened compared to the conventional method for judging the agglomeration image by natural sedimentation, and the time required for the measurement is shortened to several tens of minutes to enable the rapid measurement. . Further, the detection sensitivity can be greatly improved as compared with the conventional method, which greatly contributes to the measurement using the agglutination reaction of the antigen.

[0012]

EXAMPLES The present invention will be specifically described below with reference to examples, but the examples of the present invention are not limited thereto.

Example 1 Detection of Vibrio cholerae enterotoxin 10 μl in a 0.1 W / V% solution of latex particles having a diameter of 0.8 μm
Prepare a suspension of 0.15 W / V% latex particles with a diluent (phosphate buffered saline containing 0.5% bovine serum albumin) after mixing equal amounts of g / ml anti-Cholera enterotoxin antibody. This was used as the antibody solid phase latex solution. The purified Vibrio cholerae enterotoxin (100 ng / ml) was used as a sample and serially diluted 2-fold with a diluent. After adding 25 μl of this diluted solution to a microtiter plate (type V), 25 μl of an antibody solid phase latex solution was added, and the contents of the wells were mixed with a microplate mixer. Seal the microtiter plate with a microplate seal and immediately
Centrifugation was carried out for 10 minutes at 0 rotation to allow the latex particles to settle into a pellet in the center of the well. After the centrifugation, the microplate was tilted to about 70 ° C. and allowed to stand for 5 minutes, and the shape change of the pellet was visually observed. As a control, a reverse passive Lattex agglutination reaction was performed using the same material, and the determination was performed by spontaneous sedimentation.

The results are shown in Table 1. The criteria for determination are as shown below. Method of the present invention- : Dropping in a tear shape ±: Most of the pellet remains, but a slight tearing is observed +: The pellet remains as it is Control method-: No aggregation is observed Thing: Slightly aggregated +: Aggregated

In the method of the present invention, the time required from the start of the test to the judgment was about 25 minutes. In the conventional method, the time required from the start of the test to the determination was 18 hours. Further, as is clear from Table 1, the detection sensitivity (about 0.39 ng / ml) by the method of the present invention was four times higher than that by the conventional method (about 1.56 ng / ml).

Claims (1)

[Claims] 1. In a method for measuring an antigen using an agglutination reaction, carrier particles having an antibody corresponding to the antigen to be measured solid-phased are reacted with a sample, and the reaction product is forcibly precipitated by centrifugation to obtain A method for measuring an antigen, which comprises applying a force to the obtained sediment in a direction different from the sedimentation direction during centrifugation to measure the change in shape.