JPS61280570A - Novel analysis by electrophoresis - Google Patents

Abstract

PURPOSE:To make it possible to measure the component in a specimen from the difference of the migration patterns of a specimen set as a control one and one obtained by reacting said specimen with a specific reaction reagent with a short time with high accuracy by a small amount of a reagent, by simultaneously and respectively performing the electrophoresis of both specimens. CONSTITUTION:A reagent selected from an antigen, an antibody, coloring matter, polysaccharides lipid and a composite thereof is preliminarily included in a chip and the whole is made preservable by freeze drying. A part of the specimen from a living body is used as a reference as it is and the reagent is added to the other part thereof to be reacted with the component in the specimen. The control specimen and the reacted specimen are respectively subjected to the electrophoresis of an agarose gel for example to measure the respective migration patterns of the control specimen and the reacted specimen. In the analysis of an antigen, a monoclonal antibody is labelled with enzyme, a radioactive isotope or a fluorescent substance to enable the easy measurement of the patterns. By this method, rapid and accurate analysis is enabled with high sensitivity.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an analysis method by electrophoresis, and in particular to an analysis method using an affinity reagent that reacts with a component of a sample often used in immunological research and clinical tests. Iro: Concerning an analysis method using electrophoresis.

[Prior Art] In recent years, electrophoresis has been frequently used for protein analysis. 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 system can be divided into moving interface electrophoresis and zone electrophoresis.

In interfacial electrophoresis, an interface is formed in advance between a sample and a solvent layered thereon, 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 bands.

When a support is used, it is called support zone electrophoresis.
The purpose of the support is to stabilize the zone of separated components, and materials include:

(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 or powders, and are praised for their high sample retention and separation ability.

(3) There is polyacrylamide gel, which has a molecular sieving effect, and is recognized to have excellent separation ability because it moves according to molecular weight and has little diffusion of solutes.

In this way, in zone electrophoresis, a lot depends on the properties of the support, but as a method for analyzing the migrated zones, protein-specific staining is performed after electrophoresis, and the position and strength of the stained zones are determined. Confirmation is performed using the naked eye or 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., & Wiliams, C.A., combined the above electrophoresis with an immunological in-gel antigen-antibody precipitation reaction to perform a highly qualitative analysis using immunological specificity. made it possible and published the so-called immunoelectrophoresis 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 simultaneously diffuse through the gel, forming immunological precipitation lines at positions corresponding to each component. This is a method for analyzing specific components. However, during leverage analysis, it takes 10 to 50 hours for diffusion in the gel, and expensive affinity reagents such as antibodies are required at 0.02 m
This is a very difficult method, requiring 1 to 0.2 ml.

During this immunoelectrophoresis, if monoclonal antibodies are used in the groove, no immunological precipitation lines are created and determination by electrophoresis is impossible.

Other methods that combine electrophoresis and immunological methods include (1) rocket immunoelectrophoresis, (2) cross-immunoelectrophoresis, and (3) immunofixation.

(+) Method (2) requires relatively large amounts of affinity assays such as antibodies.
After mixing the drug with agarose to create a gel, a sample containing the antigen is placed at the origin and electrophoresis is performed, and an immunological precipitate is formed in the shape of a rocket or mountain according to the migration of the antigen. This method analyzes specific components based on their size.

(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, and the ponds are flushed out with saline to make the determination.

All of these are M during or after electrophoresis.
Immunoreactions or reactions with affinity substances were performed in an aerophoresis gel to identify components in a sample. Therefore, the method was to add a large amount of an antibody that reacts with a certain component to a gel over a wide range, thereby distinguishing a component with a positive reaction from a surrounding region with a negative reaction.

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

(1) It is uneconomical because expensive affinity reagents must be added in excess of what is really needed.

(2) The time required for the reaction is long because it includes the time for the affinity reagent and reagent F↓ to diffuse in the gel.

(3) Monoclonal antibodies do not form immunological precipitation lines with trace components in the corresponding sample during immunoelectrophoresis, making qualitative analysis impossible.

(4) When analyzing the weight components in serum materials, it is difficult to analyze because albumin and γ globulin, which are present in large amounts in serum, obscure bands of trace components around them.

An object of the present invention is to provide an economical, simple, and sensitive electrophoretic analysis method that can be performed in a short time and eliminates these drawbacks.

[Means for Solving the Problems] In order to achieve the object, the present invention is comprised of the following methods.

That is, first, the sample is divided into two parts, which are 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 the sample is added to sample B, and both are added to an electrophoresis gel. After electrophoresis, gel The sample is stained, decolorized, and dried, and the pattern of migration that appears is identified with the naked eye or with a densitometer.

Comparing the patterns of sample A and sample B, sample B
In this pattern, the spot of the component that specifically reacted with the affinity reagent is immunologically precipitated at the origin position, making migration impossible and missing, or when the amount of affinity reagent is small. , 12 minutes are missing, so the staining density is attenuated and shows a clear difference to the control sample.

Furthermore, when a monoclonal antibody is used as an affinity reagent, a clear difference in band position occurs compared to sample A as a control.

From these facts, the target component can be identified.

In the present invention, there is no problem as long as the component in the sample and the affinity reagent are reacted in a container commonly used for immunological reactions, such as a test tube, vial, spitz, or microplate. However, considering the amount of affinity reagent and operation time, it is preferable to carry out the reaction in a chip used for adding the sample to the gel for electrophoresis.

That is, a sample is filled 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, expensive affinity reagents do not need to be added to a large area, including the unreacted halves, and do not adhere to the reaction container.
Since nothing is wasted, it can be carried out with extremely small amounts.

Furthermore, there is no need for time for these to diffuse and react in the gel, resulting in the advantage that the time required to obtain results is extremely short.

II! used in the present invention! As Wasei Waraku Reagent 1, (
1) Animal and plant cells, microorganisms, or components and their products, such as Staphylococcus A protein; (2) Protein trade, such as human hemoglobin, and their complexes; (3) Hormones, drugs, and their products. Complex (4) Antigen against antibodies present in blood, brain and spinal fluid (5) Various antibodies (6) The above-mentioned substances include radioisotopes, 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 IeIs, stabilizers such as amino acids, preservatives such as sodium nitride, and the like may be added.

The chip containing the affinity reagent used in the present invention is a tubular one made of glass, plastic, stainless steel, etc., and is preferably one that can be attached to the tip of a microsyringe or micropipette. Furthermore, in order to incorporate an affinity reagent, a tip may be attached to the tip of a microsyringe or a microbed, and a certain amount of the affinity reagent solution may be aspirated.

In addition, in order to store the chip containing the affinity reagent,
Place it in a water vapor impermeable bag made of plastic, metal foil, etc.
Seal and refrigerate or freeze. Preferably, the affinity reagent solution is sucked into the chip and then 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 the reaction in the microchip to proceed rapidly, with the affinity reagent being in a solution state. The reaction time is shorter than that built into the chip.

In the present invention, since the amount of affinity reagent is constant, it may be necessary to dilute the sample as appropriate depending on the content of the component in the sample.

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) To determine the class and type of M component of myeloma serum,
Add original serum to the origin of agarose gel electrophoresis No. 1 to 0.
．． 00In+I, 0.60 times diluted serum to No-2.
For 001ml and NO-3, 0.001ml of 60-fold diluted serum was sucked into the freeze-dried chip after suctioning the pre-purified IgG antibody and reacting for 1 minute.
Similarly, sample No. 4 which had affinity absorption of I and gG was
Samples affinity-absorbed with gA antibody, NI) 5 were similarly treated with 1
A sample affinity-absorbed with an 8M antibody, a sample similarly affinity-absorbed with an antibody to No. 6, and a sample affinity-absorbed with a λ antibody to No. 7 were added, and electrophoresis was performed at a DC voltage of 200 μ for 60 minutes. , fixed with trichloroacetic acid and alcohol, then stained with Comangie brilliant blue and dried with warm air to create a clear blue migration pattern. Since defects in IgA in No. 4 and Ni in No. 6 were detected from the pattern, the M component was determined to be of IgA type.

The amount of antibody used was 0.005ml, and the time required was 2 hours.

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

First, make a thin layer of agarose gel and place origin No. 1 and 2.
, 3, 4, and 5 D. Add 0.002++ I of each of the original serum and perform electrophoresis for 60 minutes at a DC voltage of 200 V. After electrophoresis, grooves N0-1, 2, and 3 were made parallel to the direction of electrophoresis. ,4
．． 5 0.051 anti-1gG, anti-1gA, anti-1.1 (M
After adding each anti-λ antibody to the anti-antibody and leaving it at room temperature for 24 hours, an immunological precipitation line finally appeared. , the M component was determined to be type +8A.Compared to Comparative Example (1), Example (1) used 50 times the amount of antibody used, 12 times the usage time, and 11 times the required amount of sample.

Example (2) In order to determine the IgG subclass of the M component of IgG myeloma serum, serum was added to origin No. 1 of an agarose gel.
Add 100 times diluted serum to 2 ml, No-2 at 0.002
ml, 0.002 ml of anti-13Gl monoclonal antibody purified by affinity chromatography to NO-3 was aspirated, and 0.0 ml of 100-fold diluted serum was added to the freeze-dried chip.
A sample in which 02ml was aspirated and IgG1 was affinity-absorbed, N
Similarly, o-4 contained a sample affinity-absorbed with anti-13G4,
To NO-3, a sample that had been similarly affinity-absorbed with the A protein of the grape-shaped bulb was added, electrophoresed at a DC voltage of 200 μm for 60 minutes, fixed with trichloroacetic acid and alcohol, and then stained with Comangie brilliant blue. Then dry with warm air. Defects in the NO-3 band were read from this electrophoresis pattern. Generally, A protein reacts with IgG1, 2.4, but this sample did not react with anti-1gG+, 4, so the IgG subclass of the M component was determined to be lgc2.

A table for determining the subclasses identified in this manner is shown below. (Positive reaction: +, negative reaction: -) Comparative Example (2) Similarly to Example (2), when identifying the +gc subclass of the M component of IgG myeloma serum, anti-1
Inhaling the 3G monoclonal antibody in solution form,
After 30 minutes, it was added to the gel and electrophoresis was performed. As a result, the affinity absorption is incomplete, and therefore the electrophoretic migration pattern does not show a clear difference in mobility.
Only a slight attenuation was observed.

Comparative Example (3) Similarly to Example (2), conventional immunoelectrophoresis was performed using a commercially available monoclonal antibody to identify the subclass of the M component of 1gG bone marrow and lung serum. First, make KN of agarose gel and use origin No. 1, 2, 3, 4.
Add 0.002 ml of sample serum to the sample, and after electrophoresis, fill grooves No. 1, 2, 3, and 4 parallel to the direction of electrophoresis.
51 monoclonal antibodies, anti-13G+, anti-13G2
.

I added anti-1 gG3 and anti-18G4 and left it at room temperature. After 24 hours, I expected to detect an immunological precipitation line, but I could not find any pattern due to precipitation on the gel surface. , the subclass could not be identified.

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. As a result, affinity absorption of albumin in the sample was performed, and in the electrophoretic pattern, albumin disappeared and serum amyloid A protein, a 1Mfi component, which existed electrophoretically close to the sample, clearly appeared.

Comparative Example 4) An experiment similar to Example (3) was conducted. However, in this case, a chip that did not contain albumin antibodies was used.

Therefore, the trace component, the band of serum 7 myoprotein, is
It was difficult to identify because it was strongly influenced by albumin, which has the highest content in serum, and was obscured.

Example (4) There are three M components in the γ globulin region of serum, and these
It was determined whether it was Moon GG. As a preliminary test, a dilution series of commercially available peroxidase-labeled IgG was prepared and the titer was assayed, and then a chip containing 0,001 ml of peroxidase-labeled IgG antibody diluted 8 times was prepared. First, sample sail 0011 was added to the origin No. 1 of an agarose gel for electrophoresis. Next, 10 times diluted sample 0.00
1 so 1 was added to NO-2. Next, the same sample was placed on a chip containing a labeled antibody and added to origin No. 3. After electrophoresing this, it was immersed in peroxidase substrate iα and colored purple with 4 chloror:11 naphthol. N
In O-3, two bands corresponding to the M component were colored.

As a result, two of the three bands in the sample are IgG
It was confirmed that it was the M component.

Example (5) Analysis of α+AT that acts as a defense against inflammation in serum,
We tried a method of affinity absorption of the sample in a small hole with a volume of 110.05 ml in a microplate.

First, divide the sample into two and put sample A into hole No.1.
Test NB was prepared by freeze-drying α+AT antibody in advance.
-2 and reacted for 1 minute, and then electrophoresed both samples using Aga's gel. Sample A, which was used as a control, showed a pattern with a clear band at the electrophoretic α1 position. , the reacted sample B showed a pattern in which a band was missing at the α1 position, and the band that appeared in the sample was
It was possible to identify it as α+AT.

[Effects of the Invention] As is clear from the above results, when the analysis method of the present invention is used, not only the commonly used purified antibodies but also the
Conventionally, antigens and antibodies do not form precipitates even when they react, making it possible to perform experiments using monoclonal antibodies and protein A, for which immunological analysis was incomplete, making it possible to analyze unknown samples and more accurately. Analysis is now possible.

That is, the present invention solves this problem by affinity-absorbing a sample in a small container such as a chip containing an affinity reagent and then performing electrophoresis to detect the differences between the reactant and the components before absorption. We solved the problem by analyzing the differences in patterns, and at the same time, we devised an economical, short-time, and high-sensitivity analysis method, making it useful not only in the field of immunology but also in the fields of clinical testing and biochemistry. It is very useful as an analysis method to increase the accuracy of information in research and research.

Claims (7)

[Claims] (1) In the electrophoresis method, a sample is divided into two parts in advance, one sample is left as a control, the other is reacted with a specifically reactive reagent, and then each of these is subjected to electrophoresis to create a mixture of both. An analysis method using electrophoresis, characterized in that the above-mentioned components that have reacted are identified based on differences in migration patterns. (2) Claim (1) characterized in that the above reaction is carried out in a chip for adding a sample for electrophoresis.
Analysis method using electrophoresis. (3) An analysis method by electrophoresis according to claim (2), characterized in that an addition chip containing a reagent in advance is used. (4) The electrophoretic analysis method according to claim (3), wherein the reagent is contained in a chip in a freeze-dried state. (5) The electrophoretic analysis method according to claim (3), wherein the reagent is selected from antigens, antibodies, dyes, polysaccharides, lipids, and complexes thereof. (6) The electrophoretic analysis method according to claim (5), wherein the antibody is monoclonal. (7) The electrophoretic analysis method according to claim (5), wherein the reagent is labeled with an enzyme, a radioisotope, a fluorescent substance, or the like.