KR20120026728A - Enzyme activity assay using two stage immunochromatography - Google Patents

Abstract

PURPOSE: An enzyme activity assay using lateral flow immunochromatogrphy is provided to simply measure enzyme activity without special equipment and to save measuring time. CONSTITUTION: An enzyme activity assay using lateral flow immunochromatography comprises a step of detecting a conjugate by immunochromatography. The conjugate contains a tag-attached enzyme substrate and an antibody conjugate which specifically binds to marker modified by enzyme substrates. The antibody conjugate is prepared by conjugating with an antibody, gold colloid, latex, fluorescent material, luminescent material or radioactive material.

Description

Enzyme Activity Assay Using Two Stage Immunochromatography

The present invention relates to a method for measuring the activity of an enzyme present in a biological sample. More specifically, the activity of an enzyme that changes the three-dimensional structure of a substrate among enzymes present in blood or body fluids of humans or animals is characterized by immunochromatography at this stage. The present invention relates to a method for measuring enzyme activity measured using chromatography.

For example, protein kinase (Protein Kinase), which is present in a wide range of living organisms, is an enzyme that attaches a phosphate group to a protein to change an inactive protein into an active state. It plays an important role in signal transduction.

The various kinds of protein kinase itself in the living body is a macromolecular enzyme (Holoenzyme) composed of several subunits, and its catalysis is phosphorylation of the target protein regardless of its type or structure. Attaches a phosphate group to a specific amino acid of a protein of interest.

Therefore, the measurement of protein kinase activity can be made by checking whether a phosphate group is attached to a specific amino acid of a protein or polypeptide of interest and determining how much of the target protein, ie, substrate, is phosphorylated at a unit time. .

Such protein phosphorylation enzyme activity is conventionally measured by a radiometric method or a tag-attached substrate, which is a method of measuring the amount of radioactivity remaining after the enzymatic reaction using an enzyme substrate having a radioisotope attached thereto. Enzyme Immunoassay is used, and Enzyme Immunoassay or Radioactive Immunoassay, which is a method using antibodies specific for a specific protein kinase, is used as a method for measuring the presence of protein kinase itself. have.

Recently, immunochromatography (Lateral Flow) has been used to easily search for a specific analyte.This method uses a pair of antibodies that bind to the analyte and sandwich. It is applied to adsorption membranes, synthetic resins, etc., and uses a colored substance such as colloidal gold as a labeling material to determine the presence of the substance to be measured within minutes. For example, it is etched in the case of a home pregnancy test reagent. The presence of pregnancy hormone in the urine is indicated on a measuring instrument using a pair of antibodies that bind to the hormonal hormone (Human Chorionic Gonadotrophin) and colloidal gold bound to one of these antibodies to indicate pregnancy.

The conventional method of measuring the activity of protein kinase using the radioactive isotope of the above method provides a relatively accurate measurement results, but requires attention to the handling of the radioactive material and the problem that the experimental procedure is complicated.

In addition, in the case of enzyme immunoassay using a tagged substrate, the experiment takes a long time and also requires the careful attention of the investigator throughout the experiment process, which makes the experiment difficult.

In addition, the conventional measuring method as described above requires a special equipment for most of the measurement experiments and also requires a place for the installation of such equipment, so the process of collecting, storing, and transporting the sample to be measured is essential. Will be.

However, most bioenzymes are characterized by the loss of activity in a short time when they are taken out of the specimen and out of the biological system. Especially, many enzymes, such as protein kinase, are enzymatically unstable in vitro. When the conventional measurement method as described above occurs a problem that is a big obstacle in accurately measuring the activity of protein kinase.

On the other hand, conventional immunochromatography (Lateral Flow Immunochromatography) has the advantages of in situ and rapidity, but the measurement must be included in the sample and a relatively large amount of material must be present in order to measure the have.

The present invention includes an enzyme substrate (Tag) attached to the label and an antibody conjugate (Antibody conjugate) that specifically binds to the label modified by the enzymatic reaction (Antibody conjugate) and the antibody is immunologically bound to the substrate It is an object of the present invention to provide an immunochromatographic enzyme activity assay, characterized in that the conjugate formed to be detected by immunochromatography to measure enzyme activity.

The conventional technique of measuring and diagnosing analyte in an unknown sample using immunochromatography mainly measures the presence or absence of a substance through an antibody that directly recognizes the structure of the analyte to be measured. The activity of the enzyme was not measured.

Traditionally, the activity of enzymes has been used in biochemical quantitative methods using chemical reagents, and immunologically is possible only through methods that require special equipment and long, cumbersome procedures, such as radiometric or enzyme immunoassay. did.

The present invention provides a simple method for measuring enzymatic activity without special equipment such as that used in the conventional method in the field. When using the enzymatic activity measurement method using this step immunochromatography of the present invention, Of course, the cost savings are not only significant, but also when the experiment is carried out using a given report material, it is possible to obtain a very sensitive quantitative result.

The present invention includes an enzyme substrate (Tag) attached to the label and an antibody conjugate (Antibody conjugate) that specifically binds to the label modified by the enzymatic reaction (Antibody conjugate) and the antibody is immunologically bound to the substrate It is an object of the present invention to provide an immunochromatographic enzyme activity assay of this step, characterized in that the enzyme formed by detecting a conjugate formed by immunochromatography is measured.

At this time, the detection material is used, such as gold colloid, latex, fluorescent material, light emitting material or radioactive material.

Hereinafter, the implementation of the enzyme activity measurement method using the abnormal immunochromatography of the present invention will be described in detail.

Biologically, enzyme reactions occur by binding an enzyme specific for a substrate to a specific enzyme reaction site of the substrate.

In some enzyme reactions, the enzyme acts on the substrate, and the substrate remains in a form in which a specific substance is bound.

Samples to be used in the enzyme reaction step should be as fresh as possible in order not to lower the activity of the enzyme, and liquid samples are preferred unless there is a specific reason.

Depending on the type of enzyme to be measured, the sample needs to be processed in advance. For example, when measuring enzyme activity in serum only for enzymes present simultaneously in animal serum and blood cells, preventing the measurement of accurate enzyme activity by intracellular enzymes released by the destruction of blood cells. In order to separate the blood cells, such interference should be eliminated by centrifugation of the sample or removal using a special filter (Blood Seperator, Millipore).

In the case of using a non-liquid sample, it is preferable to use the sample after pulverizing and dissolving the sample in an appropriate manner so that the enzyme can react smoothly in the reaction solution.

Enzyme substrate may be a substrate that is purely isolated from the organism or synthesized substrate, and more preferably using a synthetic peptide having high specificity to a specific enzyme. For example, cAMP-dependent protein kinase, a serine / threonine kinase, has a high specificity for this enzyme, Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly). The activity of protein kinase A can be specifically measured by using a substrate such as Myelin Basic Protein Fragment 4-14. Can be measured specifically. In the case of protein tyrosine kinase (Protein Tyrosine Kinase) can be specifically measured activity using synthetic peptides, including tyrosine.

Methods of attaching a tag to an enzyme substrate are known in the art. For example, a method of attaching biotin to the alpha amino group at the amino terminus may be used to maintain the activity of the short peptide as an enzyme substrate. (Selo, I., et al . (1996). Preferential labeling of α-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays. J. Immunol . Methods 199 : 127-38.)

The reaction product of the enzyme can be captured using an antibody, more preferably a monoclonal antibody, specific for the enzyme reaction product. For example, Serine / Threonine kinase attaches a phosphate group to Serine or Threonine residues. Such monoclonal antibodies that specifically recognize phosphorylated Serine and Threonine can be produced by methods known in the art. When other structural forms of the substrate are modified by enzymes, monoclonal antibodies that specifically recognize the modified structural forms can be used to capture the enzyme reaction product.

The tag attached to the enzyme substrate can be captured using a substance that specifically binds to the tag, more preferably an antibody or a bioactive substance. For example, when biotin is used as a tag, an antibody, avidin, or streptavidin, which specifically bind to biotin, may be used.

Antibodies or bioactive proteins that specifically recognize and bind tags should be conjugated with reporter materials that will ultimately enable measurable responses. The report material may be conjugated by a method known in the art using colloidal gold, resin latex, fluorescent materials, radioactive materials, and the like.

Techniques for immobilizing antibodies on solid phases are known in the art. In general, high molecular polymers such as nitrocellulose are widely used in the stationary phase. Antibodies are immobilized to the membrane of such special polymers through ionic bonds, hydrophobic bonds, hydrogen bonds, and the like. By direct surface coating and drying.

Antigen-antibody reactions in membranes are recognized and measured by reporter materials. Colloidal gold or polystyrene latex is widely used for qualitative recognition by naked eyes. It is widely used in quantitative analysis and such measuring methods are already well known in the art.

Hereinafter, specific examples of the enzyme activity measurement method using the immunochromatography of the present invention will be described in detail.

Example 1 This example provides a preparation of a reaction solution for the activation of cAMP-dependent protein kinase in an unknown sample and a method for preparing an immunochromatography strip to capture a phosphorylated substrate by the action of this enzyme. It is about. Enzyme reaction buffer (Reaction Buffer) was prepared in the composition of 40mM TrisHCl (pH7.4), 10mM MgCl2, 1mM ATP. At this time, 60 μM Biotinylated Kemptide (Anaspec) was added to use as a substrate for the enzyme. Considering the amount of sample and the amount of the enzyme reaction product, the amount of the final reaction solution is adjusted to 40 μl.

The conjugate that binds to the biotin tag was prepared by adsorbing Goat anti-biotin antibody (Coat) on an average of 40 nm colloidal gold. At this time, 1 ml of colloid was used per 5 μg of antibody and the conjugation time was 5 minutes on average.

Strips for enzymatic product capture were prepared by immobilizing the mouse anti-phosphoserine kemptide monoclonal antibody (Calbiochem) on a nitrocellulose membrane (Milliopre,). The monoclonal antibody was adjusted to a final concentration of 1 to 5 mg / ml, and then uniformly line-printed to an amount of 0.05 to 0.2 μl per 1 mm of nitrocellulose membrane. After application, the nitrocellulose membrane was left to dry for 1 hour at 40-50 ° C. and a relative humidity of 15% or less to help fix the antibody. The dried nitrocellulose membrane was blocked with 1% BSA (1% Bovine Serum Albumin in D.W.) solution to minimize reactivity with other materials. The blocked membrane was left for 2 hours or more under the above conditions and completely dried again to be used for the experiment. At this time, the anti mouse antibody was fixed to the downstream of the anti-phosphoserine antibody to confirm the normal progress of the experiment. Anti-mouse antibody was diluted to a final concentration of 1 ~ 5mg / ml and then uniformly applied so that the amount of 0.1 ~ 0.2μl per 1mm nitrocellulose membrane.

The upper part of the nitro cellulose membrane is 20 mm long and made of synthetic fibers and the width of the nitro cellulose membrane is overlapped with the membrane by about 1 mm. The conjugate was applied in an amount of 3-10 μl. The synthetic fiber pad coated with the conjugate was dried at 20 ° C. and 15% RH, and then attached to the nitrocellulose membrane. Finally, an absorbent pad was attached to the opposite end of the nitrocellulose membrane to absorb the reaction solution passing through the membrane. The strip was completed.

Example 2 This example relates to a method for measuring the activity of enzymes contained in a sample using the reaction system and strip prepared in Example 1). The enzyme solution was prepared by dissolving Cα (PKA Cα, Sigma), a catalytic subunit of cAMP-dependent protein kinase, in DW to 5u / ml. 10 μl of the enzyme solution thus prepared was added to the reaction solution prepared in Example 1 so that the final volume of the reaction solution was 50 μl to make the reaction solution 1, and then reacted at room temperature (20-25 ° C.) for 30-60 minutes. Meanwhile, for the negative control, a reaction solution in which 10 μl of distilled water was added to the same reaction solution was made and reacted under the same conditions, thereby making Reaction Solution 2. After the reaction, 50 μl of all of the reaction solution 1 and 2 were respectively administered to the terminal portions of the synthetic fiber pads of the two test strips made in Example 1. The administered reaction solution is moved to the pad by capillary action to meet the conjugate, and the reaction solution mixed with the conjugate moves the nitrocellulose membrane so that the line of the anti-phosphoserine antibody and the line of the mouse anti-chlorine antibody Pass through the parts in turn. The line appearing 10 minutes after the reaction solution was visually observed to confirm that the strip having the reaction solution 1 showed the same color line as the red colloidal deposit in the region where the anti-phosphoserine antibody was fixed. It was confirmed that there was no change in the strip administered with liquid 2. This result is well supported by the present invention as demonstrating that the line appearing at the site where the anti-phosphoserine antibody was immobilized was caused by the product of the enzymatic reaction.

Claims (2)

Enzyme substrate attached with a label (Tag) and an antibody conjugate (Antibody conjugate) that specifically binds to the label material modified by the enzymatic reaction and the conjugate formed by the antibody immunologically bonded to the substrate This step immunochromatography enzyme activity measurement method characterized in that by detecting the enzyme activity by immunochromatography.
The method of claim 1, wherein the antibody conjugate is prepared by conjugating an antibody with gold colloid, latex, fluorescent material, luminescent material or radioactive material.