KR100976327B1 - Method and apparatus for determining human chorionic gonadotropin levels - Google Patents

Abstract

The present invention relates to a method for detecting quasi-quantitative analysis of a concentration of human chorionic stimulating hormone (hCG) in urine and a quasi-quantitative detector device therefor as a supplementary role in diagnosing the stage of pregnancy. It includes five immersion strips with dipping ends. The results are shown by the coloring of the two bands in the transparent portion of the immersion bands. One band is coated with a reagent such as a hCG antibody and the other is coated with a reagent such as goat / rabbit polyclonal antibody against monoclonal antibodies that bind to a monoclonal antibody. The combination of colors indicated on the bands provides the test results.

Human chorionic stimulating hormone, antibody gold conjugate, test band, control band

Description

Quasi-quantitative analysis and detection method of human chorionic stimulating hormone concentration in urine and its detector mechanism {METHOD AND APPARATUS FOR DETERMINING HUMAN CHORIONIC GONADOTROPIN LEVELS}

The present invention relates to techniques for determining whether a person has a particular disease, health condition, illness or pain, and in particular determines the level of human chorionic gonadotropin (abbreviated as hCG). A method and apparatus for

hCG is produced during pregnancy and is produced by the cells that form the placenta that nourish the fertilized egg after the sperm and egg are fertilized and implanted on the wall of the uterus. hCG can be detected initially by blood testing about 11 days after implantation and urinalysis on 12-14 days. In general, the concentration of hCG doubles every 72 hours, peaks at 8-11 weeks of gestation, and then drops during pregnancy.

Table 1 below shows that the concentration of hCG in blood or urine is proportional to the gestational weeks since the last menstrual cycle (LMP).

Therefore, the detected concentration of hCG can be used as an aid in determining the gestational age and the state according to the various stages of pregnancy. For example, low hCG concentrations may indicate spontaneous miscarriage, bright ovum, or ectopic pregnancy. High concentrations of hCG may indicate molar pregnancy or multiple pregnancy.

There are many commercially available pregnancy diagnostic kits on the market. However, existing products only provide a "yes" or "no" answer to whether or not hCG was detected. No additional information is provided regarding the various concentrations of detected hCG. Doctors often use the concentration of hCG to monitor the course of pregnancy. However, there are currently no products that provide point care technology (POC) for providing convenient, inexpensive, and prompt medical care.

For example, to determine the concentration of hCG, blood must be drawn and standard laboratory tests called the ELISA hCG test performed. The results show the actual concentration of hCG in mIU / ml in blood, but include and require an invasive procedure called blood collection. To obtain the concentration of hCG, the collected blood must be transferred to a specialized laboratory where the test technician performs the test. Generally, the laboratory is far from the hospital where the blood is collected. The ELISA hCG test only takes about an hour, but additional time is needed for the test results to be sent back to the hospital. Therefore, at least one day is usually required to receive the test results. Another drawback is the cost of testing in specialized laboratories that require additional labor and materials.

Pregnancy weeks can be determined using transvaginal ultrasound examination. Choking ultrasound can be used to identify at least a gestational sac at about 5 weeks of pregnancy, i.e. when the concentration of hCG reaches between 1,000 and 2,000 mIU / ml. Ultrasound technology, however, does not know the state of early pregnancy, which is the most important time to observe a normal or abnormal pregnancy.

There is currently one commercial test capable of detecting hCG concentrations of 2,000 mIU / ml or higher. This test is a Wampole immunochemical latex agglutination slide test. Doctors use this product to observe changes in hCG levels in pregnant women after abortion, assuming that the concentrations of hCGs have dropped sufficiently. Therefore, there are many variations in the sensitivity of the Wampole slide test. Moreover, the latex flocculation process of the Wampole slide test is slow and the precision is not sufficient for diagnostic purposes. Sensitivity varies from 1,000 mIU / ml to 3,000 mIU / ml. Since the Wampole slide test is for detecting high concentrations of hCG, it provides only one hCG concentration without any semi-quantitative information.

As mentioned above with respect to the disadvantages of the current hCG concentration measurement test, there is a need for hCG test instrument overcoming the disadvantages of the current test, more simple to use, easy to measure, cheap, accurate.

The present invention relates to a detection method and detector tool capable of quickly analyzing various hCG concentrations in urine samples. This detection method and apparatus is based on antigen-antibody reactions. The detection kit consists of multiple bands with several bands assembled in a cassette, with backing sub-strips, absorbent materials sub-strips, windows or transparent sections. Cover sub-strip, a portion of an antibody gold conjugate, and the like. Auxiliary bands are covered with a short section of adsorbent materials exposed to the dipping tentacle. Inside the window are the letters "T" and "C" that indicate the location of the test and control bands, respectively.

The test band is coated with hCG antibody. The control band is coated with antibody reagents such as goat / rabbit polyclonal antibodies. Antibody gold conjugates bind to hCG antigens in urine samples.

In this test, the immersion in the urine causes the urine sample to move through the adsorbent material and pass through the portion of the antibody gold conjugate and several bands. There are two different techniques for detecting 25mIU / ml, 100mIU / ml, or 500mIU / ml, and for detecting 2,000mIU / ml and 10,000mIU / ml. At the first three concentrations of hCG, i.e., when the concentration of hCG in the samples is greater than 25mI / ml, 100mIU / ml, 500mIU / ml, respectively, the antibody gold conjugate is associated with the hCG antigens in the urine sample in a relevant window. Will bind, bind to the hCG antibody in the test band (“T” line), and appear crimson. At 2,000mIU / ml and 10,000mIU / ml concentrations, when the hCG concentrations in the sample are above 2,000mIU / ml and 10,000mIU / ml, respectively, antibody gold conjugate binding will be inhibited in the "T" line. It will not develop color. However, if the sample crosses the control band, the antibody gold conjugate will appear scarlet in the "C" line because it binds to the goat / rabbit polyclonal antibodies coated on that band. This color change in the "C" line means that the reagents and test materials are working properly. If there is no color change in the control band, it may indicate that there is not enough urine in the sample.

Other aspects and advantages of the present invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings in the detailed description of the invention.

According to the above problem solving means, the present invention can quickly detect various concentrations of hCG in the urine sample based on the color change displayed on the bands of the multiple bands and the combination result, and as a result, Overcoming the shortcomings of simple diagnostic tests, it is easier to use steps proportional to gestational weeks, easier to measure, cheaper, and accurate hCG concentration testing.

The present invention is a detection method and apparatus capable of quickly detecting various concentrations of hCG in a urine sample, and is based on an antigen-antibody reaction. To be specific, multiple test bands were used to measure various hCG concentrations. Each test strip has multiple result zones that include a test zone and a control zone. Each test strip is coated with reagents such as hCG antibodies in the test zone and goat / rabbit polyclonal antibodies against monoclonal antibodies in the control zone. Antibody gold conjugates bind to hCG antigens in urine and then to antibodies in the test zone.

In the hCG test, the urine sample is delivered to the test strip by dipping the test strip into the urine sample. It travels through the adsorbent material of the test strip, passes through the antibody gold conjugate region, and reaches the resulting region coated with antibody material. There are two major chemical reactions in the five test strips. The first detects 25mIU / ml, 100mIU / ml and 500mIU / ml, and the second detects 2,000mIU / ml and 10,000mIU / ml. At three low concentrations of hCG, i.e., when the concentration of hCG in the sample is higher than 25mI / ml, 100mIU / ml, and 500mIU / ml, respectively, the antibody gold conjugate binds to the hCG antigen in the urine sample and then in the test zone. It binds with hCG antibody and shows crimson.

At 2,000 mIU / ml and 10,000 mIU / ml, when the hCG concentration in the sample is higher than 2,000 mIU / ml or 10,000 mIU / ml, respectively, the hCG antibodies in the test area are blocked by high concentrations of hCG antigens, resulting in antibody gold conjugates. gold conjugate) will not appear crimson in the test area. However, the control zone is colored, which means that the urine sample has passed to the control zone. The reaction is based on the binding of the antibody / antibody gold conjugate to the goat / rabbit polyclonal antibody coated in the control zone, and the change in color indicates that the reagent and test zone were exposed to urine samples and functioning normally. If there is no change in color in the control zone, then there is not enough sample to react with the reagents in the control zone.

If the hCG concentration in the sample is lower than 2,000 mIU / ml or 10,000 mIU / ml, respectively, both the test zone and the control zone of the test strip will be crimson / red. That is, the test is negative indicating that the hCG concentration in urine is lower than 2,000 mIU / ml or 10,000 mIU / ml. Color change in only the control zone indicates that this test band was exposed to samples with positive or hCG concentrations greater than 2,000 mIU / ml or 10,000 mIU / ml. Finally, if both the test and control zones did not change color, this test indicates that the test is invalid for various reasons, such as insufficient amount of sample.

The components of the test strips and their contents are as follows. Here, the test bands show that the predetermined human chorionic gonadotropin concentration is divided into an affinity portion that detects 2,000 mIU / ml or less and an inhibition portion that detects 2,000 mIU / ml or more. have.

The components of the 25 mIU / ml test strip and their contents were selected from the group consisting of goat / rabbit polyclonal antibody, 30 ug / test --- control band that binds to monoclonal antibody; Goat anti human chorionic stimulating hormone antibody, 50.2 ug / test --- test band; And Anti-hCG antibody-Colloidal Gold Conjugate, 0.43 ug / test --- glass fiber.

The components of the 100 mIU / ml test strip and its content were selected from the group consisting of goat / rabbit polyclonal antibody, 30 ug / test --- control band; Goat anti human chorionic stimulating hormone antibody, 46 ug / test --- test band; And Anti-hCG antibody-Colloidal Gold Conjugate, 0.39 ug / test --- glass fiber.

The components of the 500 mIU / ml test strip and their contents were selected from the group consisting of goat / rabbit polyclonal antibody, 30 ug / test --- control band; Goat anti human chorionic stimulating hormone antibody, 38 ug / test --- test band; And Anti-hCG antibody-Colloidal Gold Conjugate, 0.39 ug / test --- glass fiber.

The components of the 2,000 mIU / ml test strip and their contents were selected from goat / rabbit polyclonal antibody, 54 ug / test --- control band; Goat anti human chorionic stimulating hormone antibody, 18 ug / test --- test band; And Anti-hCG antibody-Colloidal Gold Conjugate, 0.23 ug / test --- glass fiber.

The components of the 10,000 mIU / ml test strip and their contents were selected from the group consisting of goat / rabbit polyclonal antibody, 54 ug / test --- control band; Goat anti human chorionic stimulating hormone antibody, 9.8 ug / test --- test band; And Anti-hCG antibody-Colloidal Gold Conjugate, 0.08 ug / test --- glass fiber.

In other words, in the affinity part where the human chorionic stimulating hormone concentration is detected at 2,000 mIU / ml or less, the amount of antibody gold conjugate and T decreases as the human chorionic stimulating hormone concentration in the urine sample increases. As the amount of antibody gold conjugate decreases to 100, 500mIU / ml, it can be seen that the color of the test band changes only when the concentration of human chorionic stimulating hormone is high.

In addition, in the inhibition portion where the concentration of human chorionic stimulating hormone is detected to be 2,000 mIU / ml or more, some human chorionic stimulating hormone is not bound to the antibody gold conjugate while it is bound up the band. The chorionic gonadotropin first rises along the band and binds in the test band, thereby preventing the antibody gold conjugate from binding to the test band's antibody.

1 shows a sample of a cassette 10 having multiple bands 12, 14, 16, 18, 20 to embody the present invention. Multiple bands are assembled in the cassette 10. Each strip 12, 14, 16, 18, 20 includes a sheath support strip, an adsorbent material strip (end), and a sheath support strip having some transparent window 22. The adsorbent band is supplemented with glass fibers with an anti-hCG antibody-colloidal gold conjugate. Below the transparent portion of the window 22 of the band is a control band containing goat / rabbit polyclonal antibody and a test band of goat anti-hCG antibody. The glass fibers with the antibody gold conjugate are placed between the adsorbent band and the shell band with the transparent window 22. Auxiliary bands are coated with a membrane along with a short portion of each auxiliary band end of the adsorbent material that is exposed to the dipping end when the tip is immersed. The window has the notation "T" and "C" inside to indicate the location of the test and control bands respectively.

The adsorbent material consists of two components, two filter papers. The first type 1 is cotton fiber with a wet strength of 70 and the second type 2 is glass fiber. The window in the cassette consists of cellulose nitrate with a hole size of 5um to 10um and a wicking rate of 90sec / 4cm. Antibody gold conjugates are absorbed by immersed and imbued samples in immersion tentacles. If the hCG concentration is 25mIU / ml, 100mIU / ml, 500mIU / ml, 2,000mIU / ml and 10,000mIU / ml or less, it binds to the hCG antibodies coated on the test band and shows the color of each test band. If hCG concentrations are higher than 25mIU / ml, 100mIU / ml, 500mIU / ml, 2,000mIU / ml, and 10,000mIU / ml, the hCG antigens in the sample may be replaced with antibodies coated on the test band in place of the antibody gold conjugate. Couple to suppress coloration of the test band. Antibodies in the control band are goat / rabbit polyclonal antibodies against mouse monoclonal antibodies.

FIG. 2 depicts multiple bands 12, 14, 16, 18, 20 of cassettes contained in urine samples. Urine samples are absorbed by the adsorbent material that attracts the sample contained in the test bands and control bands of the multiple bands. Each test band is coated with hCG antibody. Each control band is coated with reagents such as goat / rabbit polyclonal antibodies against mouse monoclonal antibodies. Antibody gold conjugates of adsorbents bind hCG antigens in urine samples. When the urine sample has been wetted, the urine travels through the adsorbent material through the antibody gold conjugate region towards the bands. If the hCG concentration in the sample is higher than 2,000 mIU / ml or 10,000 mIU / ml, the hCG antigen in the urine sample binds to the hCG antibodies in the test band and blocks the binding of the antibody gold conjugate to 2,000 mIU / ml and 10,000 mIU. / ml Suppresses the appearance of color in the window. When the urine sample passes the control band, the antibody gold conjugate binds to the reagents in the control band and expresses a scarlet band, so the control band becomes scarlet regardless of the concentration of hCG in the sample. The color change of the control band indicates that the reagents and test materials are working correctly. No color change in the control band indicates that there is not enough urine present in the sample. In one example, if the urine sample is too thin and has a certain low specific gravity, the urine may not be sufficient to color the control band.

If the hCG concentration in the urine sample is higher than 25mI / ml, 100mIU / ml, or 500mIU / ml, the antibody gold conjugate binds to the hCG antigens in the urine sample. The combination of hCG antibodies from each of the 25mIU / ml, 100mIU / ml and 500mIU / ml window test bands resulted in a scarlet appearance. As the urine sample passes the control band, the antibody gold conjugate binds to the goat / rabbit polyclonal antibodies coated on the control band, making the control band crimson regardless of the hCG concentration in the sample. The color change of the control band indicates that the reagents and test materials are working correctly. If the control band does not change color, this test is not valid because it indicates that the urine sample was not sufficient.

Figure 3 depicts a cassette showing a positive result of 25mIU / ml. Test bands of 100mIU / ml and 500mIU / ml show no color change. This indicates that the measured hCG is lower than 100mIU / ml and 500mIU / ml but higher than 25mIU / ml. Since the color of the test band of 2,000mIU / ml and 10,000mIU / ml appears, this indicates that the measured hCG concentration is lower than 2,000mIU / ml and 10,000mIU / ml. Therefore, this test shows positive at 25 mIU / ml. Note that the color of the control band on the test strip indicates that the test is valid.

4 depicts the cassette showing a negative result for measurable hCG. Test bands of 25mIU /, 100mIU / ml and 500mIU / ml are colorless. This means that the measured hCG concentration is lower than 25mIU / ml. Test bands of 2,000 mIU / ml and 10,000 mIU / ml show color. This indicates that the measured hCG concentrations are lower than 2,000 mIU / ml and 10,000 mIU / ml. This test shows that the hCG concentration is lower than 25 mIU / ml, since the appearance of color in the control band of the test band indicates a valid test.

Figure 5 depicts a cassette indicating an invalid test because no color appears in the control bands of the test bands.

6 depicts a cassette showing positive results at 100 mlU / ml. 25mIU / ml and 100mIU / ml test bands are colored. This indicates that the measured hCG concentration is at least 100 mIU / ml. However, since there is no change in color in the test band of 500mIU / ml, it means that the measured hCG is below 500mIU / ml. Colors appear in test bands of 2,000 mIU / ml and 10,000 mIU / ml. This indicates that the measured hCG concentrations are lower than 2,000 mIU / ml and 10,000 mIU / ml. Thus, this test shows a positive result at 100 mIU / ml. The color of the control band on the test strip indicates that the test is valid.

7 depicts a cassette showing a positive result at 500 mIU / ml. 25mIU / ml, 100mIU / ml and 500mIU / ml test bands are colored. This indicates that the measured hCG concentration is at least 500mIU / ml. Colors appear in the test bands of 2,000 mIU / ml and 10,000 mIU / ml. This indicates that the measured hCG concentrations are lower than 2,000 mIU / ml and 10,000 mIU / ml. Thus this test shows a positive result at 500 mIU / ml. The color of the control band on the test strip indicates that the test is valid.

8 depicts a cassette showing positive results at 2,000 mIU / ml. 25mIU / ml, 100mIU / ml and 500mIU / ml test bands are colored. This indicates that the measured hCG concentration is at least 500mIU / ml. There is no change in color in the test band of 2,000 mIU / ml, but only in the test band of 10,000 mIU / ml. This indicates that the measured hCG concentration is at least 2,000 mIU / ml but lower than 10,000 mIU / ml. Thus this test shows a positive result at 2,000 mIU / ml. The color of the control band on the test strip indicates that the test is valid.

9 depicts a cassette showing positive results at 10,000 mIU / ml. 25mIU / ml, 100mIU / ml and 500mIU / ml test bands are colored. This indicates that the measured hCG concentration is at least 500mIU / ml. No color appears in the test bands of 2,000 mIU / ml and 10,000 mIU / ml. This indicates that the measured hCG concentrations are at least 2,000 mIU / ml and 10,000 mIU / ml. This test therefore shows a positive result at a hCG concentration of at least 10,000 mIU / ml. The color of the control band on the test strip indicates that the test is valid.

While a detailed description including the specification of the invention at this stage, it should be recognized that various changes and modifications are technically possible without departing from the spirit of the invention. Furthermore, the scope of the present invention is only as defined by the following claims.

1 shows a sample of a cassette having multiple bands to embody the present invention.

2 depicts multiple bands of a cassette contained in a urine sample.

Figure 3 depicts a cassette showing a positive result of 25mIU / ml.

4 depicts a cassette showing negative results for measurable hCGs.

5 depicts a cassette indicating an invalid test.

6 depicts a cassette showing positive results at 100 mlU / ml.

7 depicts a cassette showing a positive result at 500 mIU / ml.

8 depicts a cassette showing positive results at 2,000 mIU / ml.

9 depicts a cassette showing positive results at 10,000 mIU / ml.

Claims (13)

Using multiple bands comprising regions coated with antibody gold conjugates that react with human chorionic gonadotropin in urine, and having multiple result zones coated with different kinds of antibody material for which antibody-antigen reactions occur; Forming a plurality of bands in accordance with a predetermined human chorionic gonadotropin concentration, and combining a plurality of bands each formed such that the concentrations are in a multi-stage ascending order to form a test cassette; Contacting the multiple bands with urine to absorb a urine sample; Human chorionic gonadotropin of the urine sample reacts with the antibody gold conjugates of the multiple bands to form an antibody-antigen complex, and the process of color change due to the antibody-antigen reaction in multiple result regions, respectively A quasi-quantitative detection method of human chorionic stimulating hormone concentration in the urine, comprising the step of qualitatively detecting the human chorionic stimulating hormone concentration in multiple steps based on the combination. 2. The goat or rabbit polyclonal antibody of claim 1, wherein the multiple bands comprise glass fibers coated with antibody gold conjugates and the multiple result zones are bound to test bands and monoclonal antibodies coated with an anti human chorionic stimulating hormone antibody. Consisting of a control band coated with; In the step of detecting the qualitative analysis of the human chorionic gland stimulating hormone concentration, When the concentration of human chorionic stimulating hormone in the urine sample reaches a predetermined human chorionic stimulating hormone concentration, the antibody-antigen complex is combined with an anti-human chorionic stimulating hormone antibody to change the color of the test band; , Quasi-quantitative analysis of human chorionic stimulating hormone concentrations in urine characterized by the fact that the antibody gold conjugate is exposed to a urine sample and the color of the control band changes by binding to a goat or rabbit polyclonal antibody against a monoclonal antibody. Detection method. The method of claim 2, wherein the multiple bands comprise an affinity portion for detecting a predetermined human chorionic gonadotropin concentration of 2,000 mIU / ml or less and an inhibition for detecting 2,000 mIU / ml or more. Quasi-quantitative detection method of human chorionic stimulating hormone concentration in urine, characterized in that divided into parts). The method of claim 3, wherein the multiple bands, Human chorionic stimulating hormone concentration in urine characterized by a predetermined human chorionic stimulating hormone concentration of 25mIU / ml, 100mIU / ml and at least five, such as 500mIU / ml and 2,000mIU / ml and 10,000mIU / ml Quasi-quantitative analysis detection method. The method according to claim 4, wherein when the human chorionic stimulating hormone concentration of the urine sample is 25 mIU / ml or more, the process of changing the first test band to crimson, The process by which the first and second test bands turn scarlet when the concentration of human chorionic stimulating hormone in the urine sample is 100 mIU / ml or more, Quasi-quantitative analysis of human chorionic stimulating hormone concentrations in urine including the process of changing the first, second and third test bands to crimson when the concentration of human chorionic stimulating hormone in urine samples is above 500 mIU / ml Detection method. The process according to claim 5, wherein the color of the fourth and fifth test bands turns crimson when the concentration of human chorionic stimulating hormone in the urine sample is lower than 2,000 mIU / ml. The process in which the color of the fourth test band does not change when the concentration of human chorionic stimulating hormone in the urine sample is 2,000 mIU / ml or more, A method for qualitative quantitative analysis of human chorionic stimulating hormone concentration in urine comprising a process in which the color of the fifth test band does not change when the human chorionic stimulating hormone concentration of the urine sample is 10,000 mIU / ml or more. 7. The method according to claim 6, wherein the color of the first, second, third, fourth, and fifth control bands is changed to crimson. Embedding antibody gold conjugates in multiple test bands, and applying a test band of human chorionic stimulating hormone antibody to each portion of the test bands to form multiple bands; In the course of immersing multiple bands in a urine sample, the human chorionic gland in the urine comprising flowing the sample through the antibody gold conjugate to the human chorionic stimulating stimulating hormone antibody in the test band to produce multiple results. Stimulating hormone concentration qualitative quantitative analysis detection method. 9. The method of claim 8, further comprising applying a control band of goat or rabbit polyclonal antibody to each portion of the plurality of test bands to form multiple bands; A method for quasi-quantitative detection of human chorionic stimulating hormone concentration in urine further comprising a wet step of flowing a sample to a goat or rabbit polyclonal antibody in the course of immersing multiple bands in a urine sample. Multiple bands comprising antibody gold conjugates that react with human chorionic gonadotropin in urine and have multiple result zones coated with different types of antibody material for which antibody-antigen reactions occur; The multiple bands are formed in accordance with a predetermined human chorionic stimulating hormone concentration, but the human chorionic gonadotropin in the urine including a test cassette formed by combining a plurality of multiple bands each formed so that the concentrations are multisequential. Concentration quasi quantitative detector. The quasi-quantitative detector of human chorionic stimulating hormone concentration in urine according to claim 10, wherein the cassette comprises a transparent window made of cellulose nitrate. The method of claim 10 wherein the multiple bands, Human chorionic hair in urine, comprising a test band formed by applying an anti-human chorionic stimulating hormone antibody to each part and a control band formed by applying a goat or rabbit polyclonal antibody to each other part SAM stimulating hormone concentration quasi-quantitative detector. The method of claim 12, wherein the multiple bands, Human chorionic stimulating hormone in urine characterized by a predetermined human chorionic stimulating hormone concentration of 25mIU / ml, 100mIU / ml and 500mIU / ml and at least five, such as 2,000mIU / ml and 10,000mIU / ml Concentration quasi quantitative detector.

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