KR20070117239A - Apparatus and method for detecting allergen - Google Patents

Abstract

An apparatus and a method for detecting allergen are provided to diagnose allergy easily and quickly by utilizing an immuno-chromatography using a matrix column and an electro-chemical using electrode reaction together. An apparatus for detecting allergen comprises an automatic electric potential control device(1), a recorder(2), and a plurality of electrodes(3). The apparatus for detecting allergen detects serotonin by measuring electro-chemical reaction of a sample(7). The automatic electric potential control device controls electric potentials from 0 to 1.0V automatically. The apparatus for detecting allergen detects serotonin with current value measured at 0.3V at which oxidation occurs.

Description

Allergen Detection Device and Detection Method {APPARATUS AND METHOD FOR DETECTING ALLERGEN}

1 is a diagram showing an electrode response to serotonin in the blood.

2 is a view showing an allergen detection device.

3 is a view showing the arrangement of the microelectrodes of the allergen detecting apparatus.

4 is a view showing the change in the current value after the injection of the antigenic material for hay fever.

5 is a diagram illustrating a detection principle of an antigen-antibody complex using a matrix column.

Figure 6 is a diagram showing the isoelectric point of each antigen antibody complex, Fc-IgG antibody, BSA-histamine.

7 is a graph showing the ion exchange capacity test results of the matrix column.

8 is a diagram showing the electrochemical output value according to the change in the histamine concentration.

9 shows the reproducibility of histamine for electrochemical detection.

10 shows a correlation between radioimmunoassay and On-chip type electrochemical assay.

The present invention provides a self-care type allergen detection apparatus and method for diagnosing allergy by using an electrochemical detection method using an electrode reaction, an immunochromatography method using a matrix column, and an electrochemical method using an electrode reaction.

Conventional allergy tests have a prick test, and a recent diagnostic method is a kind of blood test, and a method of making a diagnosis by measuring the amount of IgE in serum is used.

The Prick test is a conventional method of using an eyedropper to drop an antigenic substance into an arm and stabbing it with a needle, and then examining the allergic reaction, which is an allergic reaction to an allergen. However, this method is classified as very dangerous in some people because of the possibility of shock from hypersensitivity.

When a more accurate diagnosis is required, individual hospitals are sometimes referred to a testing institution (large hospitals, such as general hospitals), which is very time consuming, which makes it difficult to expect a faster diagnosis or prescription. This method is a type of blood test that measures by measuring the amount of IgE in the serum. When allergens enter our body, an IgE antibody called immunoglobulin E is produced. Since the IgE antibody is produced more than the normal person, the concentration of IgE antibody in the blood can be detected relatively higher than the normal person. This is one of the most frequently used tests for the diagnosis of allergies, but there are problems with this method, and IgE antibody concentrations are not enough to accurately diagnose allergies. In some cases, even though the diagnosis is negative depending on the result of the IgE antibody concentration, the reaction may show a similar symptom indicating positive, and it takes about 3 to 5 days until the final diagnosis, which is expensive.

Therefore, in order to solve the above problems of the prior art, the present invention uses an electrochemical detection method using an electrode reaction, an immunochromatography method using a matrix column, and an electrochemical detection method using an electrode reaction. Technically original and innovative method compared to the conventional method is to provide an apparatus and method for detecting the allergens simple and quick operation.

One of the methods used to diagnose allergies is prick test, which has been classified as a very dangerous method because of the possibility of shock due to hypersensitivity reactions. It is a blood test to measure the concentration of IgE antibody in serum. The diagnostic method is expensive and takes 3 to 5 days, so it takes a lot of time for diagnosis and prescription. Therefore, the electrochemical detection method using an electrode reaction, immunochromatography method and electrochemical method to detect allergens from the present invention. The present invention provides a self-care type allergen detection device and method capable of a simpler and faster diagnosis for a patient suffering from allergies by using a chemical method in combination.

This object is achieved by the present invention, and in accordance with an aspect of the present invention, the allergen detection device of the present invention includes an automatic potential control device for automatically adjusting the potential; A recorder for displaying the measured result; And detecting serotonin by measuring an electrode chemical reaction of a sample composed of a plurality of electrodes.

And the electrode is characterized in that the potential is automatically adjusted to 0 ~ 1.0V for serotonin detection.

And serotonin is detected by the current value measured at 0.3V oxidation reaction.

In addition, the electrode is made of gold (Au), composed of 65 pairs of microbands (microband), the width of the microband is 8 ~ 12μm, the interval is 4 ~ 65μm, the length of the electrode is 1.8 ~ 2.2mm It characterized in that it comprises a fine electrode made.

In the present invention, a method using an immunochromatography method and an electrochemical detection method using a matrix column,

(B) contacting the sample causing the disease to be tested with a labeled antibody specific for the disease to be tested with the sample under conditions permitting an antigen-antibody reaction between the sample and the labeled antibody;

(2) adsorbing a labeled antibody that has not reacted to the disease to be tested by immunochromatography from the reaction solution obtained in the above reaction: and

질병 Efficacy of the disease (antigen) marker to be eluted, characterized in that the detection of the disease substance by a process comprising a step of detecting the signal from the antibody complex electrochemically.

Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment of the present invention.

Patients who begin to have allergies will have histamine, cholin, serotonin, noirin, trimethylamine, tyramine and phenylethylamine in their blood over time. (phemylethyl-amine), tryptamine (tryptamine), etc. are secreted, and allergens can be diagnosed by detecting the secreted allergens. To this end, two methods were applied: electrochemical detection using electrode reaction, immunochromatography and electrochemical detection using electrode reaction.

Allergens to propose a detection method from the present invention are serotonin and histamine.

First, electrochemical detection was used to detect serotonin. As a result of investigating the electrode response to serotonin in the blood at a constant speed (10mV / min) from 0 to 1.0V per unit time, the general form of Cyclic Voltammonogram as shown in FIG.

1 is a diagram showing the electrode response to serotonin secreted in the blood, a general graph of the form that means that the electrons emitted at 0.3V is transferred to the electrochemical oxidation occurred by measuring the current value at that time serotonin concentration Quantification of is possible. In order to quantitatively measure serotonin concentration, it is possible to derive correlation with current values for various concentrations. The device used for such an electrode reaction was manufactured in a simplified manner as shown in FIG. 2, and the microelectrode of the allergen detection device in FIG. 2 is shown in FIG. 3.

Then, it was investigated through the clinical trial whether the serotonin can be detected electrochemically with the simple allergen detection system of FIG. 2. Therefore, the concentrations of IgE antibodies generally analyzed for allergy patients and the values detected according to a simple electrochemical allergen detection method from the present invention are analyzed for allergy sufferers and healthy people, respectively. Table 1 is shown. In FIG. 4, the change in the detected current value was almost insignificant in a healthy person, and the value was small enough to be negligible.

Table 1 Electrochemical Detection and IgE Antibody Concentration

Detection target Electrochemical Allergen Detection (nA) Amount of IgE antibody (UA / ml) diagnosis A person with hay fever 6.8 2.16 + healthy person 0.6 0.16 －

However, in case of allergy (polludosis), the current value is rapidly increased compared to a healthy person over time. IgE antibody concentration analysis was used to determine whether these currents are really allergic. As a result, in Table 1, a person suffering from hay fever had a positive reaction (+, 2.16 UA / ml), and a healthy person had a negative reaction.

Then, a clinical test was conducted on 17 persons in their 20s and 30s to see if the results were correct. More specifically, pollinosis was examined. The results obtained by comparing the current values and IgE antibodies by the electrochemical detection method are shown in Table 2. > Is shown.

In Group A, the electrochemical detection method showed a positive agreement between the current value and the IgE antibody concentration.

In the case of Group B, positive IgE antibody concentration was detected. However, the detection of current value by electrochemical method showed one of two current values corresponding to negative reaction, but the diagnostic result was positive. This result indicates that some people do not release serotonin in the blood despite allergic reaction to hay fever.

TABLE 2 Current value and IgE antibody concentration by electrochemical method for hay fever

Gender (age) Current value (nA) Amount of IgE Antibody Symptoms A group B group C group D group Male (35) Male (29) Male (27) Female (29) Female (33) Female (25) Male (23) Female (36) Male (28) Male (25) Male (22) Male (23) Male ( 24) Male (24) Female (23) Male (24) Female (26) 3.9 14.6 4.5 3.2 8.7 4.1 9.5 0.7 -1.2 -0.2 1.1 -0.7 0.3 -0.7 -4.8 3.1 2.3 11.1 10.3 10.7 7.4 4.7 2.2 1.3 39.0 19.5 0.34 <0.34 <0.34 <0.34 <0.34 <0.34 <0.34 <0.34 < + + + + + + + + +--------+, ± +, ±

* ±: pseudopositive sympyoms

In case of Group C, all of the symptoms of the electrochemical detection method and the result of IgE antibody concentration showed a negative result. In the case of Group D, the IgE antibody concentration was less than 0.34, which was negative, whereas the current value by the electrochemical detection method was positive, and the patient was found to have a positive response and expressed as pseudo-positive symptoms. This can be explained by the fact that some people do not feel allergies even if they have antibodies.

Therefore, only three of the 17 patients had an error in diagnosis by the electrochemical detection method of allergens through the results of Groups A ~ D, which resulted in sensitivity exceeding 83%. The electrochemical detection of serotonin was very effective.

In order to detect histamine using the allergen detection system of FIG. 2, the same test was performed with serotonin. However, the oxidation potential of histamine was in the range of 0.35 to 0.40V, and it was very difficult to selectively detect only histamine because of various substances detected in this range.

Therefore, we tried to theoretically establish whether histamine can be detected by using immunochromatography using isoelectric point and electrochemical detection using electrode reaction.

A method of separating histamine and an electrochemical detection method of the separated material by immunochromatography using a matrix column coated with a credit card sized ion exchange resin is shown in FIGS. 5 and 6.

First, a mixed sample containing an antibody (Fc-IgG antibody) immobilized with ferrocene and an antigen-antibody complex produced in step (I) in which an antigen is incubated in an immune reactor under a predetermined condition, that is, in (I) Antibody antibody complexes, unreacted Fc-IgG antibodies, and unreacted antigens are injected into the matrix column of step (II) coated with ion exchange resin using Buffer (pH 6.5). Histamine-Fc-IgG Complex is adsorbed in step (II), and BSA-Histamine and BSA-Hitamine-Fc-IgG Complex, which exhibit the characteristics of anions in step (III), are separated from the matrix column.

At this time, each isoelectric point is shown in <6>, and it is a result which can be understood that it is separated by the isoelectric point. Then, <Fig. 5> (IV) was investigated by electrochemical detection method using a matrix column coated with an ion exchange resin in order to use the immunochromatography how the concentration of histamine and the current value change.

Fc-IgG showing the characteristics of the cation whether the cation exchange resin was not coated on the matrix column of step (II) (A) and (B). Fc-IgG antibody concentration was examined at the portion discharged when the antibody was injected, and the result is shown in FIG. 7.

The peaks of the uncoated matrix column (A) resulted in very little adsorption of the cations and the separation resulted. In the case of the coated (B), Fc-IgG was discharged from the cation exchange resin. Since no peak indicating the antibody concentration was detected, the matrix column coated with the cation exchange resin was very effective.

From these results, we analyzed the current value according to the change of histamine concentration by competition method. This is a method of detecting histamine by what to read electrochemically when other substances are present in addition to the antigen-antibody complex, and the measurement result was satisfactory as shown in FIG.

In addition, when the histamine secreted into the blood of the patient is detected electrochemically in the allergic patients and healthy people with hay fever whether the reproducibility of the detection results are shown in <Figure 9>.

In FIG. 9, healthy people showed higher reproducibility in the vicinity of 35nA as shown in FIG. 8, and good results were reproduced in the vicinity of 12nA in the hay fever. In the same way, <Table 3> shows histamine from blood for 6 people including 5 healthy and 5 allergic patients. Indicated.

Table 3 Detection of Histamine in Patients with hay fever

Gender (age) Current value (nA) Symptoms Female (23) Female (24) Female (23) Male (23) Female (31) Male (25) * 22.0 16.0 13.6 24.8 12.0 35.2 + + + ± +-

Ferrocene-IgG concentration: 3 μg / ml, BSA-Histamine concentration: 40 μg / ml

       ± people with similar symptoms, who do not feel even if they have antibodies

      * Sample of healthy people

If the histamine detected in this way is well correlated with the radioimmunoassay measured by the test institute, more reliable data can be evaluated, and the results are analyzed and shown in FIG. 10.

As a result, the data examined by the two methods were found to be highly correlated, and compared with the existing detection results by using the immunochromatography method using the matrix column and the detection method with the chip to enable the electrochemical detection method. As shown in Table 4, the results are very reliable.

<Table 4> Comparison with existing detection methods

Technology Detection method Detection target Pretreatment Required time to detection Sensitivity Specificity Jongrak's technology Radioimmunoassay MAST IgE antibody detection limit in blood 1.0 U / ml U Hours 75% 80% CAPRAST Detection limit of IgE antibody in blood 0.35 U / ml U Hours 86% 92% Non-radioactive immunoassay Enzyme Immunoassay Detection limit of IgE antibody in blood 0.80 U / ml U Hours 84% 91% Fluorescence Immunoassay Detection limit of IgE antibody in blood 0.50 U / ml U Hours 85% 93% This technology Electrochemical measurement Detection limit of IgE antibody in blood 50 ng / ml radish 15 minutes 83% 93%

* MAST: Multi Antigen Simultaneous Test

* CAPRAST: Cap Radio-immuno Sorbent Test

Here, comparing histamine detection technique with the conventional technique using an immunochromatography method using an on-chip type electrochemical measurement method using a matrix column, the detection method according to the present invention can measure up to 50 ng / ml without pretreatment. It was found that measurement was possible within a short time (15 minutes).

These results will be a good opportunity to recognize the need for a faster and easier allergy diagnosis system. The reason is that the existing histamine measuring device is large and difficult to use in a private hospital, and it takes 13 to 17 days for histamine and 3 to 5 days for IgE antibody until diagnosis results are obtained. There was a difficulty of delaying.

However, with the introduction of the diagnostic system according to the present invention, it is expected that a faster and simpler construction of a detection system will be required in the future. In addition, since the causative agent causing allergies may provide a plurality of causes such as eggs, beans, milk, and flour, a multi-allergen detection system is required because there is a need to evaluate each.

In the present invention, the electrochemical detection method of serotonin is a system that can detect any allergen if only antigen is present. Allergens can be quickly detected by electrochemical methods.

In addition, the use of two methods, such as immunochromatography and electrochemical detection using electrode reactions, can be used to diagnose not only serotonin and histamine, but also the causes of various diseases. Any kind of protein can detect any substance. As a result, any disease can detect specific proteins secreted from the blood of a person with the disease, which means that the disease can be diagnosed.

The present invention made as described above can diagnose allergies more quickly and conveniently by using an electrochemical detection method using an electrode reaction, an immunochromatography method using a matrix column, and a method using an electrochemical detection method using an electrode reaction. As for the allergen detection method of the new concept, a matrix column downsized to the size of a credit card was manufactured to enable allergen detection.

From these results, access to the mobile micro-diagnosis system is expected to be facilitated, it is possible to provide an in-home medical diagnosis system, and to be presented as an optimized on-chip multi-detection system.

In addition, it will be possible to build a system of cooperation between personal biometric information and medical devices, enabling customized medical-based technology. The technology presented in the present invention is a useful invention that the market in the food industry and environmental measurement field is very expected in addition to the medical diagnostic field because the basic protocol is universal.

Claims (6)

An automatic potential control device for automatically adjusting the potential; A recorder for displaying the measured result; And an allergen detection device configured to detect serotonin by measuring an electrochemical reaction of a sample composed of a plurality of electrodes. The allergen detection device according to claim 1, wherein the potential is automatically regulated from 0 to 1.0V. 2. The simplified allergen detection device according to claim 1, wherein serotonin is detected by a current value measured at 0.3 V in which an oxidation reaction has occurred. According to claim 1, wherein the electrode is made of gold (Au), consisting of 65 pairs of microbands (microband), the width of the microband is 8 ~ 12μm, the interval is 4 ~ 65μm, the length of the electrode is 1.8 An allergen detection device, characterized in that it comprises a fine electrode consisting of ~ 2.2mm. In the method using an immunochromatography method using a matrix column and an electrochemical detection method in combination, First step: contacting the sample causing the disease to be tested, a labeled antibody specific for the disease to be tested, and a condition in which an antigen-antibody reaction between the sample and the labeled antibody is possible; Second step: adsorbing a labeled antibody unreacted to the disease to be tested by immunochromatography from the reaction solution obtained in the reaction: and Third step: Allergen detection method characterized in that the detection of the diseased substance quantitatively by a step comprising the step of electrochemically detecting the signal from the test target disease (antigen) marker-antibody complex to be eluted. 6. The allergen detection method according to claim 5, wherein the electrochemical detection of the third step detects histamine by a current value measured on the basis that the potential is automatically adjusted to 0 to 1.0V.

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