JPS61280569A - Chip for adding specimen and its preparation - Google Patents

Abstract

PURPOSE:To make it possible to simply analyze protein or a compound thereof within a short time, in electrophoretic analysis, by including a compatible reagent specifically reacted with the component in a specimen. CONSTITUTION:A specimen is divided into specimens A, B and, after the compatible reagent specifically reacting with the specimen B was added to the specimen B to be reacted therewith, both specimens are added to an electrophoretic gel to perform electrophoresis and the gel is subsequently stained, decolored and dried to discriminate the developed migration pattern by the naked eye or a densitometer. When the patterns of the specimens A, B are compared, in the pattern of the specimen B, the spot of the component specifically reacted with the compatible reagent is immunologically sedimented at the position of an origin and migration becomes impossible to generate omission and, therefore, the staining concn. thereof is attenuated to perfectly discriminate the specimen B from the specimen A.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a chip used in an electrophoretic analysis method, and particularly to a chip containing an affinity reagent that reacts with a component in a sample.

[Prior art] In recent years, 'i'I!' has been used for protein analysis. Aerophoresis is widely used. This principle utilizes the physicochemical phenomenon that when charged particles are placed in an electric field, the distances traveled by the particles differ depending on the difference in their charges.

This electrophoresis method can be divided into moving interface electrophoresis and zone electrophoresis.

In interface pneumophoresis, an interface is formed in advance between a sample and a solvent layered on top of the interface, and the movement of solute molecules in the solution is observed in an electric field. On the other hand, zone electrophoresis is a method in which each component is fractionated and separated into bands by applying voltage to a sample added in the form of bands. When a support is used, it is called support zone electrophoresis, and the purpose of the support is to stabilize the zone of separated components, and the following materials are available:

(1) Typical porous materials include filter paper and cellulose acetate, which are often used in clinical tests because they are inexpensive and easy to operate.

(2) Agar and starch are often used as gels and powders, and they have a high sample retention capacity and high separation ability.

(3) Polyacrylamide gel has a molecular sieving effect, and is recognized to have excellent separation ability due to its migration according to molecular weight and low solute diffusion.

In this way, in zone electrophoresis, although there are many differences depending on the properties of the support, protein-specific staining is performed after electrophoresis as a method for analyzing the migrated zones, and the position and strength of the stained zones can be observed with the naked eye or is confirmed using optical methods, but confirmation of separation is often extremely difficult due to the large number of overlaps.

In order to solve this drawback, in 1953, Grabar
, P. & Williams, C.A. combined the above-mentioned electrophoresis with in-gel antigen-antibody precipitation to enable highly sensitive qualitative analysis utilizing immunological specificity. Presented electrophoresis method.

In this method, a small hole is made in a thin agar gel, a sample containing the antigen is added thereto, electrophoresis is first performed to develop and separate each component in the sample, and then the electrophoresis is performed parallel to the direction of electrophoresis. When antibodies are placed in the opened grooves and left for a certain period of time, the sample and antibodies will diffuse through the gel, forming immunological precipitation lines at positions corresponding to each component. , is a method for analyzing specific components. However, during leverage analysis, it takes about 10 to 50 hours for diffusion in the gel, and expensive affinity reagents such as antibodies require only 0.02ml of
This is a very difficult method, requiring approximately 0.2 ml of water.

Other combinations of WI electrophoresis and immunological methods include (1) Rocket Immunization M, pneumophoresis, 2) cross-immunoelectrophoresis, and (3) immunofixation.

Methods (1) and (2) involve mixing a relatively large amount of affinity reagents such as antibodies with agarose to create a gel, then placing a sample containing the antigen at the origin and performing electrophoresis, following the migration of the antigen. This is a method in which immunological precipitates are formed in the shape of rockets or mountains, and specific components can be analyzed based on their size.

Method (3) is a method in which after zone electrophoresis, the gel is immersed in antibodies, and only the specific zones that react with the antibodies are left fixed as precipitates, while the rest are flushed out with saline to perform the determination.

In all of these methods, components in a sample are identified by performing an immunoreaction or a reaction with an affinity substance in an electrophoresis gel during or after electrophoresis. Therefore, a method was used to distinguish a specific component with a positive reaction from surrounding areas with a negative reaction by adding a large number of antibodies or affinity reagents that react with a specific component to the gel over a wide range.

[Problems to be Solved by the Invention] As described above, conventional electrophoresis has the following drawbacks.

(1) Expensive affinity reagents must be added in excess of the amount truly needed, which is uneconomical.

(2) These antibodies are usually commercially available in a lyophilized, frozen, or refrigerated state, and when used, lyophilized products are made into a liquid by adding a certain amount of water, or frozen and refrigerated products are brought to room temperature. After that, you must take out only the amount you need and use it.
This is extremely troublesome and time-consuming work.

(3) Antibodies in the state described in (2) often lose their activity or become spoiled, and are wasted unless they are used in their entirety twice.

(4) The time required for the reaction is long because it includes time for the affinity reagent and sample to diffuse through the gel.

(5) In the case of immunoelectrophoresis using monoclonal antibodies, no immunological precipitation lines are created, making qualitative analysis impossible.

(6) When analyzing trace components in serum materials - Due to albumin and γ globulin present in large amounts in serum, the electrophoretic pattern of surrounding trace components is obscured, making analysis difficult.

[Object of the Invention] The present invention was made in view of these drawbacks, and its purpose is to develop electrophoresis and immunoreaction that are economical, simple, and capable of analyzing proteins and their compounds in a short time. An object of the present invention is to provide an addition chip incorporating an affinity reagent for use in a new analysis method, and a method for manufacturing the same.

[Means for Solving the Problems] The present invention is a chip that can be applied to the novel electrophoretic analysis method described below, which allows analysis to be completed in a short time and is easy to operate.

That is, the sample is divided into parts and designated as samples A and B. A is left unchanged as it is used as a control.

Next, an affinity reagent that specifically reacts with the components in sample B is added to sample B, and both are added to an electrophoresis gel and electrophoresed. After staining, decolorizing, and drying, the pattern of migration that appears is identified with the naked eye or with a densitometer.

Then, when comparing the pattern of sample B to the sample, sample B
In this pattern, the spot of the component that specifically reacted with the affinity reagent may be immunologically precipitated at the origin position, making migration impossible and missing, or if the amount of affinity reagent is small, a portion of the spot may be lost. Due to the lack of , the staining density is attenuated, showing a clear difference from the control sample A.

Furthermore, when a monoclonal antibody is used as a f0 affinity reagent,
There is a clear difference in the position of the band compared to sample A used as a control.

From these facts, the target component can be identified.

In the present invention, the location where the components in the sample and the affinity reagent are reacted is
There is no problem as long as it is in a container commonly used for this type of reaction, such as a test tube, vial, spitz, or microplate. However, considering the amount of affinity reagent and operation time, it is optimal to use the chip of the present invention.

That is, it is preferable to carry out the reaction in a chip used for adding the sample to the gel. That is, a sample is aspirated into a chip containing a certain amount of affinity reagent in advance, reacted with components in the sample, and then added to an electrophoresis gel. In this way, there is no need to add expensive affinity reagents to a large area, including areas where the reaction is negative, and there is no need to waste the affinity reagents by adhering them to the reaction container, so you can save a lot of money. It is possible to implement it. In addition, there is no need for time for these to diffuse and react in the support, resulting in an advantage that the time required to obtain results is extremely short.

Examples of affinity reagents used in the present invention include (1) animal and plant cells, microorganisms, or constituents and their products, such as the A protein of grape-like bulbs, and (2) proteins such as human hemoglobin. , their complexes (3) Hormones, drugs, etc. and their complexes (4) Antigens against antibodies present in blood, brain and cerebrospinal fluid (5) Various antibodies (6) The above include radioisotopes and fluorescent reagents. ,
Some are labeled with enzymes, etc.

These may be used alone or as a mixture of a plurality of them. Alternatively, various additives such as stabilizers such as sugars and amino acids, preservatives such as sodium nitride, and dyes for identifying affinity reagents may be added to these.

The chip containing the affinity reagent used in the present invention may be made of any material, such as glass, plastic, or stainless steel, as long as it does not react with the sample or the affinity reagent, but it is preferably made of a material to which these substances do not easily adhere. Some polyolefin compounds such as polypropylene, polyethylene, and polyfluorinated ethylene are suitable.

Further, the tip used in the present invention may have a capillary shape, but from the viewpoint of operability, it is desirable that the tip be of a shape that can be attached to the tip of a microsyringe or micropipette. Therefore, it goes without saying that known conical tips for micropipettes can be used. However, it is preferable that the base is thick enough to be attached to a micropipette or microsyringe, and the tip has a capillary shape with a length of at least 101 or more. Using a tip with such a shape makes it easy to add a sample to a support such as a gel. The outer diameter of the capillary part is 0.5 mm to 1,01111 mm.
1. The inner diameter is preferably about 3 to 0.5 mm.

To incorporate an affinity reagent, attach the tip to the tip of a microsyringe or micropipette and aspirate a certain amount of the affinity reagent solution, or if the tip is for a micropipette, use its conical shape. It is also possible to fill by dripping from the thick tip.

To store the chip containing the affinity reagent, place it in a water vapor-impermeable bag made of plastic, metal foil, etc., seal it, and refrigerate or freeze it. Preferably, after filling the chip with a solution of the affinity reagent, the chip is stored in a dry state by lyophilization or the like. This not only improves storage stability, but also allows rapid mixing and contact between the sample and the affinity reagent, allowing for even small reactions in the cup.
It proceeds rapidly and requires a shorter reaction time than if the affinity reagent was in solution and contained in the chip.

Furthermore, the freeze-drying conditions used in the present invention are as follows. That is, the temperature of the sample is between -10°C and -80°C, and the degree of vacuum is preferably 5 m+++Hg or less. All known electrophoresis materials and operations can be used in the present invention. In addition, all known methods can be used to detect differences in patterns, but among them, electrophoretic patterns are generated using enzyme reactions, radioautography, complement hemolysis reactions, etc., and these can be viewed with the naked eye or densitometry. The method of identification using a meter is particularly effective.

Examples of the present invention are described below, but these are not intended to limit the scope of the present invention.

[Example] Example (1) A capillary tube made of polytetrafluoroethylene with an inner diameter of 0.38 n+m, an outer diameter of 0.85 nua, and a length of 30 mm, with a tip shaped so that the base can be attached to a microsyringe. The purified IgG antibody was mixed with 1% glycine and 1% sucrose as antibody stabilizers, and 0.01% Bonso R for identification. 0.002 ml of this was suctioned, and 001 ml of air was further suctioned from the tip to move the antibody from the tip to the center of the chip, and it was frozen at -80°C.

After the freezing was completed, the chips were freeze-dried for 3 hours while maintaining a vacuum level of 5+mmHg to obtain the chip of the present invention.

Example (2) To determine the class and type of M component of myeloma serum,
Agarose gel? Place 0.001ml of serum at the origin of aerophoresis, No. 1, and place 60 times diluted serum at No. 2.
1. ml, NO-3 with pre-purified IgG antibody.
A sample was obtained by aspirating 0.002 ml of 60-fold diluted serum into a lyophilized chip and immunoabsorbing IgG, No. 4 was a sample similarly immunoabsorbed with IgA antibody, and No. 3 was a sample immunoabsorbing with IgA antibody. Similarly, a sample was immunoabsorbed with an 18-open antibody, and No. 6 was a sample that was similarly immunoabsorbed with an antibody.
To No. 7, a sample immunoabsorbed with the absorbed body was added, electrophoresed at a DC voltage of 200 V for 60 minutes, fixed with trichloroacetic acid and alcohol, then stained with Comangie brilliant blue, dried with warm air, Creates a clear blue migration pattern. From that pattern, No. 4 IgA and No.
Since the deletion of J(-6) was detected, the M component was determined to be IgA type.

The amount of each antibody used was 0.002 ml, and the required time was 2 hours.

Comparative example (1) The analysis of Example (1) was performed by conventional immunoelectrophoresis.

First, make an agarose gel HN, and set the origin No. 0-1.2.
, 3 and 4.5, add 0.002 Il+l of the original serum and perform electrophoresis for 60 minutes at a DC voltage of 200 V. After electrophoresis, grooves No. 1, 2, 3, and 4 made parallel to the direction of migration
, 5, 0°0511 anti-1gG, anti+gA, anti-1gM
After adding each antibody, anti-, anti-in, and leaving it at room temperature for 24 hours, an immunological precipitation line finally appeared. The M component was determined to be IgA type.

In Example (]), the amount of antibody used was 50% compared to Comparative Example (1).
It required 12 times more time, and 11 times more samples.

Example (2) In order to determine the IgG subclass of the M component of IgG bone marrow serum, serum was added to origin No. 1 of an agarose gel at O10.
Add 100 times diluted serum to 0.00ml, No-2.
2ml, 0.002ml of anti-1861 monoclonal antibody previously purified by affinity chromatography on NO-3
Aspirate 100 times diluted serum into the freeze-dried chip.
．． 002ml was aspirated and IgG1 was affinity-absorbed, No-4 was a sample that was affinity-absorbed with anti-13G4, and No-3 was a sample that was affinity-absorbed with Staphylococcus A protein. was added, electrophoresed for 60 minutes at a DC voltage of 200 V, fixed with trichloroacetic acid and alcohol,
Next, it is dyed with Comangie Brilliant Blue and dried with warm air. From this electrophoresis pattern, only NO-3 was found to be missing in the M component band. Generally, A protein is Ig
G1,2.4, but this sample reacts with anti-13G+,4
Since no reaction occurred, the IgG subclass of the M component was determined to be 1gG2.

Comparative Example (2) Similar to Example (2), I of the M component of IgG myeloma serum
When identifying the gG subclass, there is anti-1g in the chip.
The G monoclonal antibody was inhaled in the form of a solution, and 30 minutes later, it was added to the gel and electrophoresis was performed. As a result, the affinity absorption was incomplete, and therefore, no change was observed in the band in the electrophoretic pattern, making it impossible to determine the class.

Example (3) In order to analyze a trace component in a sample serum, serum amyloid A protein, electrophoretic analysis was performed in the same manner as in Example (2) using a chip containing an albumin antibody. Affinity absorption of albumin in the sample takes place, resulting in
In the electrophoretic pattern, albumin disappeared, and serum amyloid A protein, a trace component present in electrophoretic proximity, clearly appeared.

Comparative Example (3) An experiment similar to Example (2) was conducted. However, the chip used in this case does not contain albumin antibodies. Therefore, the trace component serum amyloid protein that exists in close proximity is strongly influenced by albumin and is obscured, making it difficult to identify.

Example (4) There are three M components in the γ globulin region of serum, which are I.
It was determined whether it was gG. As a preliminary test, we prepared a series of multiple dilutions of commercially available peroxidase'tRm IgG and tested its titer, and then created a chip containing 0.001ml of peroxidase-labeled 13G antibody diluted 8 times. . 0.001 ml of sample was added to origin No. 1 of an agarose gel for electrophoresis. Next, 0.001 ml of a 10-fold diluted sample was taken and added to NO-2. Next, the same sample was placed on a chip containing a labeled antibody, and origin No.
added to. After electrophoresis, the peroxidase substrate was soaked overnight and colored purple with 4-chloro-1-naphthol. In NO-3, two bands corresponding to the M component were colored. From this fact, two of the three bands in the sample
It was confirmed that the book was the M component of IgG.

[Effects of the Invention] From the above results, it is clear that when the affinity reagent of the present invention is subjected to affinity absorption using a built-in chip using freeze-drying technology and then subjected to M, pneumophoretic analysis, general In addition to purified antibodies that are commonly used, experiments using monoclonal antibodies and protein A, which do not produce precipitates even when antigens and antibodies react, and for which immunological analysis has been incomplete, are now possible. This has made it possible to analyze unknown samples and more accurately.

That is, the present invention solves this problem by performing electrophoresis after affinity absorption in a chip containing an affinity reagent and analyzing the difference in pattern between the reactant and the component before absorption. I was able to do it.

At the same time, it has been devised to be economical, to perform analysis in a short time, and with high sensitivity, so it is useful not only in the field of immunology, but also in the fields of clinical testing and biochemistry, as an analytical method that increases the accuracy of diagnostic and research information. As such, it is very useful.

Claims (7)

[Claims] (1) A chip for addition characterized by containing an affinity reagent that specifically reacts with components in a sample. (2) The additive according to claim 1, wherein the affinity reagent is a dye, polysaccharide, lipid, or a complex thereof that reacts with an antibody, an antigen, or a component in a sample. Chip. (3) The chip for addition according to claim (1), wherein the affinity reagent is a substance labeled with an enzyme, a radioisotope, a fluorescent substance, or the like. (4) The tip for addition according to claim (1), characterized in that the tip can be attached to a microsyringe or a micropipette. (5) The additive chip according to claim (1), wherein the material of the chip is a polymer of an olefin compound. (6) The base has an inner diameter that can be attached to a microsyringe or micropipette, and the tip has a length of at least 1
The chip for addition according to claim (3), characterized in that it has a shape having a capillary portion of 0 mm or more. (7) A method for producing a chip for sample addition, which comprises filling a certain amount of an affinity reagent into a chip, and then freeze-drying it and incorporating it into the chip.