KR20230088992A - A Kit for Diagnosing pregnancy, and Method for Diagnosing pregnancy Using the Same - Google Patents

Abstract

The present invention relates to a pregnancy diagnosis kit and a method for diagnosing pregnancy in animals using the same, and more specifically, to a progesterone-BSA conjugate as a test line at one end of a thin-film paper having pores of a predetermined size formed therein. ) in a predetermined area, and an assay strip having sheep-derived anti-mouse immunoglobulin (sheep anti-mouse IgG) immobilized on the other end as a control line; and an anti-progesterone mouse immunoglobulin gold-labeled conjugate to be reacted with serum or skim milk as a test sample. The pregnancy diagnosis kit of the present invention can immediately test the pregnancy of Korean cows, cows, and cows with a simple operation without any additional equipment before and after 20 days after artificial insemination. By determining pregnancy/non-pregnancy, a more accurate test can be performed.

Description

Pregnancy diagnosis kit and method for diagnosing pregnancy in animals using the same {A Kit for Diagnosing pregnancy, and Method for Diagnosing pregnancy Using the Same}

The present invention relates to a pregnancy diagnosis kit and a method for diagnosing pregnancy in animals using the same.

The estrus cycle of a normal dairy cow is 19-22 days, and on the day of estrus, the progesterone concentration in raw milk is close to 0, then gradually increases, and on the 19th day after the estrous day, the progesterone concentration decreases again to close to 0, but if the cow During pregnancy, progesterone levels do not decrease. Therefore, by taking raw milk and blood between days 19 and 22 and measuring the concentration of progesterone, it is possible to confirm whether the cow is pregnant or not pregnant.

Until the 1990s, in order to determine pregnancy/non-pregnancy of artificially inseminated dairy cows, veterinarians performed direct rectal examination 50 to 60 days after artificial insemination or breeding of dairy cows to determine pregnancy/non-pregnancy of dairy cows. judged. However, this method requires skilled artificial insemination or veterinarians, and causes a lot of economic loss due to the long idle period of cows. and the possibility of abortion could not be ruled out, and could stress the cow. In addition, since the above method can determine whether pregnancy/non-pregnancy is certain only when 50 to 60 days have elapsed after fertilization, if it is judged that pregnancy is not possible at this time, check the signs of estrus again and perform the fertilization procedure. There was a hassle to go through.

Therefore, since the 1990s, methods that can accurately and simply assess the timing of fertilization, early pregnancy, and reproductive disorders of Korean cattle or cows have been developed one after another, one example of which is a pregnancy/non-pregnancy diagnosis method using an ultrasound diagnostic device. However, since the diagnosis of pregnancy/non-pregnancy of Korean cattle is often performed by non-experts in rough fields, the ultrasound diagnostic device must be manufactured very robustly, and the equipment is large and heavy, making it difficult to apply to the field.

Subsequently, a kit was developed to diagnose pregnancy/non-pregnancy of Korean cattle by examining the concentration of progesterone in the blood using an enzyme immunoassay method. However, this diagnostic kit also required expensive analysis equipment for the test and the test process was complicated, so it was difficult for the owner to use it directly in the field.

A method of using an assay strip as a method of diagnosing pregnancy/non-pregnancy in a simple way that can compensate for such disadvantages has been reported by Laitinen et al. (Acta Chemica Scandinavia, 1996: 50: 141-145). The assay strip is A monoclonal antibody of progesterone is immobilized on a nitrocellulose membrane, a sample extracted from a cow (milk, etc.) is transferred on the membrane by capillary action, and then a progesterone-obualbumin conjugate to which colloidal gold particles are bound (hereinafter referred to as “gold particles”) on the membrane.

At this time, when a high level of progesterone is present in the sample, the progesterone in the sample first binds to the monoclonal antibody of progesterone, and the gold particles pass through without being able to bind to the monoclonal antibody of progesterone, so the color of the assay strip changes. will not do On the other hand, if progesterone is present in the sample at a low level, only a portion of the progesterone monoclonal antibody binds to progesterone in the sample, and gold particles that migrate later bind to the progesterone monoclonal antibody, so the color of the assay strip this will change Therefore, it is possible to determine whether the cow is pregnant/non-pregnant by observing the concentration of the changed color of the assay strip or by using a chromatography scanner.

However, in the above method, the sample and the gold particles must be separately moved on the assay strip to analyze whether the sample is pregnant/non-pregnant, so the operation is cumbersome, and when the cow is pregnant (ie, the amount of progesterone in the sample is high) case), there is no change in the color of the assay strip even after the diagnosis is completed, so misdiagnosis due to defects in the assay strip cannot be prevented. There is a problem in that the judgment of pregnancy/non-pregnancy should be made arbitrarily according to the observer.

As described above, in the prior art, there are cases in which the secretion of progesterone is insufficient depending on the time of pregnancy, so there are frequent cases in which pregnancy is unclearly indicated even after pregnancy. In order to solve this problem, the present invention has been made, The diagnostic kit of the present invention can diagnose pregnancy/non-pregnancy with high accuracy.

Specifically, the present invention relates to a pregnancy diagnosis kit and a method for diagnosing pregnancy in animals using the same, and more particularly, by examining the concentration of progesterone present in blood, milk, etc. Distinct species) It relates to a pregnancy test kit that can easily determine pregnancy at an early stage and a method for diagnosing pregnancy in animals using the same.

The present invention was devised to solve the above problems, and after correcting the pregnancy of Korean cattle, dairy cows, and Japanese cattle (Japanese cattle) by examining the concentration of progesterone present in blood and milk, etc., 20 It is an object of the present invention to provide a pregnancy diagnosis kit and a method for diagnosing pregnancy that can be easily determined before and after work, and which are designed to quickly and accurately test pregnancy with a simple operation. Another object of the present invention is to provide a diagnostic kit and a method for diagnosing pregnancy that can prevent misdiagnosis that may occur due to a defect in an assay strip. In addition, an object of the present invention is to provide a kit for diagnosing pregnancy and a method for diagnosing pregnancy in animals, in which a baseline is prepared so that the pregnancy state can be confirmed with high reliability.

In order to achieve the above object, the present invention coats a predetermined area of a progesterone-BSA conjugate as a test line on one end of a thin-film paper having pores of a predetermined size therein, and the other end an assay strip immobilized with anti-immunoglobulin (anti-IgG) as a control line; and an anti-progesterone immunoglobulin gold-labeled conjugate to be reacted with serum or skim milk as a test sample. Here, the anti-progesterone immunoglobulin gold-labeled conjugate (anti-progesterone IgG-gold conjugate) is preferably used after dissolving it in a phosphate buffer containing a nonionic surfactant as a lyophilisate, and the control line is derived from sheep. It is preferable that anti-mouse immunoglobulin paper (anti-mouse IgG derived from sheep) is immobilized on the thin-film paper.

The present invention is highly practical because it can easily diagnose pregnancy by measuring the color development of the concentration of progesterone, a pregnancy hormone secreted from the corpus luteum of the ovary directly related to pregnancy. Specifically, the anti-progesterone immunoglobulin Z-gold label conjugate (anti-progesterone IgG-gold conjugate) and the process of mixing the analysis sample extracted from the sample; A process of immersing the end of a progesterone-bovine serum albumin conjugate layer and a thin-film paper coated with anti-immunoglobulin (anti-IgG) at a predetermined distance from the progesterone-bovine serum albumin conjugate layer into a solution in which the analysis sample is mixed ; and comparing the intensity of color development between the progesterone-bovine serum albumin conjugate layer and the control line, and determining that the test line is pregnant when the color development level of the test line is weaker than that of the control line.

The pregnancy diagnosis kit according to the present invention is a simple method for determining pregnancy and non-pregnancy, especially pregnancy, of cows such as Korean cattle, dairy cows, and Japanese cattle (Japanese cattle) by using the concentration of progesterone present in blood and milk. can be determined early. Since the pregnancy test kit of the present invention has a control line that contrasts with the test line, it is possible to check whether or not the test strip is defective, and it is easy to determine pregnancy with high precision based on the objective criterion of comparing the concentration of the color of the control line and the test line. There are advantages to doing so.

1 is a diagram showing an assay strip constituting a pregnancy diagnosis kit according to an embodiment of the present invention.
2 is a diagram showing the principle of color development of the pregnancy test kit according to the present invention.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example

In the pregnancy diagnosis kit according to the present invention, when about 20 days have elapsed after artificial insemination on the day of estrus, the concentration of progesterone in serum, plasma, milk, etc. does not decrease in the case of pregnancy. In this case, it is to confirm whether the cow is pregnant by using the phenomenon in which the concentration of progesterone is significantly reduced, and the anti-progesterone immunoglobulin gold-gold label mixed with analysis samples such as serum, plasma, milk, etc. It consists of an anti-progesterone IgG-gold conjugate and an assay strip for visually checking the concentration of progesterone in the analysis sample. Through such a pregnancy diagnosis process, if Korean cattle are not pregnant, artificial insemination can be quickly performed again to reduce the sample's gestation period.

FIG. 1 shows an example of an analysis strip constituting a pregnancy test kit according to the present invention. As shown in FIG. 1, the analysis strip moves from one end to the other through capillary action. It consists of a thin-film paper (1) that can move to the end and a sample absorbent paper (6) that is combined with the thin-film paper (1) and functions to absorb the liquid sample that has passed through the thin-type paper (1), in the form of a strip has

The size of the assay strip is not particularly limited as long as an appropriate amount of the assay sample is transported by capillary action and the degree of reaction between the sample and the assay strip can be observed, but a size of approximately 4 mm X 50 mm is preferred. Preferably, a plurality of micropores having a diameter of 5 to 12 μm are formed on the inside of the thin-film paper 1, so that a fluid such as serum, plasma, or milk is used as an anti-progesterone immunoglobulin-gold label. It is preferable that it can be easily absorbed and moved to the thin-film paper 1 together with the bonded body.

When the diameter of the micropores is less than 5 μm, when the sample is absorbed from the surface of the paper 1 to the inside, the rate of absorption and movement by capillarity is too slow to be practical, and the diameter of the micropores is 12 μm. If it exceeds, the moving speed of the sample becomes too fast, and there is a disadvantage in that the diagnosis of pregnancy or not is inaccurate. Therefore, by appropriately adjusting the size of the micropores, it is possible to control the speed at which a component necessary for a diagnostic reaction, for example, a steroid hormone such as progesterone, passes through the test line in the sample, and the time required for diagnosis can be properly controlled. there is. The material of the thin paper 1 is not particularly limited, but is preferably made of a nitrocellulose membrane, and the sample absorbent paper 6 is preferably made of cellulose paper.

Progesterone-BSA conjugate (2) is placed on at least one side of the thin-film paper (1) at a predetermined distance from the end, preferably about 8 to 12 mm (preferably 10 mm). A predetermined area is coated as a test line, and a control line combined with an anti-immunoglobulin layer (anti-IgG: 3) is coated at a predetermined distance from the test line of the progesterone-bovine serum albumin conjugate layer (2). The anti-immunoglobulin (anti-IgG) forming the control line is preferably an anti-immunoglobulin (anti-IgG) derived from any one animal selected from the group consisting of mouse, goat and sheep, more preferably is an anti-immunoglobulin (anti-IgG) of the control line, and is an immunoglobulin derived from the same species as the immunoglobulin (IgG) of the anti-progesterone immunoglobulin gold-labeled conjugate (anti-progesterone IgG-gold conjugate). Those obtained by immunization with globulin can be used.

The anti-progesterone immunoglobulin gold-labeled conjugate constituting the pregnancy test kit according to the present invention is derived from a species that can specifically react with the anti-immunoglobulin of the control line, preferably Preferably, an anti-progesterone mouse immunoglobulin gold label conjugate is used, and a commonly available stabilizer may be included if necessary. Stabilizers used in the conjugate include, but are not limited to, bovine serum albumin, sucrose, etc., and the amount of such stabilizers varies depending on the type of stabilizer used, but is generally 1 It is preferable to use 5% to 5% by weight. The anti-progesterone immunoglobulin G-Au conjugate is prepared by freeze-drying with the addition of a stabilizer, and a ratio such as 1% Tween 20 (Hayashi Purechemical Industries Ltd., Osaka), 1% Tween 40, BSA, etc. is added to the freeze-dried conjugate. After dissolving by adding a certain amount of phosphate physiological saline buffer containing a warming surfactant, it is mixed with serum, plasma, milk, etc. of test subjects such as Korean cows and cows, and used as a pregnancy diagnostic reagent.

The function of the pregnancy test kit according to the present invention configured as described above and the principle of diagnosis of pregnancy are as follows.

First, inspection of Korean cattle and cows 19 to 21 days after artificial insemination with a drop (about 0.025 mL) of anti-progesterone immunoglobulin gold-labeled conjugate solution prepared by dissolving the lyophilized conjugate in a certain amount of buffer. A drop (0.04 mL) of a sample such as serum, plasma or milk collected from a subject is added dropwise, mixed, and then the tip of the assay strip is immersed in the obtained mixture. In this way, when the assay strip is deposited in the mixed solution, the conjugate containing the sample for analysis moves upward along the assay strip by capillarity, and passes through the progesterone-bovine serum albumin conjugate layer, which is the test line, while anti-progesterone immunoglobulin The gold-labeled conjugate binds to the progesterone-BSA conjugate layer through an antigen-antibody reaction, thereby exhibiting color development.

2 is a view for explaining the principle of color development of the pregnancy test kit according to the present invention. As shown in FIG. 2, a progesterone-bovine serum albumin conjugate layer ( 2) is formed, and while the diagnostic reagent including the anti-progesterone immunoglobulin gold-labeled conjugate 10 passes through the conjugate layer 2, the antibody-gold conjugate 10 becomes the progesterone-bovine conjugate. It reacts and binds to the serum albumin complex layer (2).

At this time, when Korean cattle are pregnant, the concentration of progesterone (20) inside the diagnostic reagent is as high as 5 ng/ml or more, so most of the anti-progesterone immunoglobulin gold-labeled conjugate (10) is progesterone present in the sample ( 20) and does not bind to the progesterone-bovine serum albumin conjugate layer 2 on the thin paper 1, so the color development of the test line is weaker than that of the control line. Conversely, in the case of non-pregnancy, since the concentration of progesterone 20 inside the diagnostic reagent decreases to 1 ng/ml or less, most of the anti-progesterone immunoglobulin G-gold conjugate 10 is progesterone on the top of the thin paper 1 - Since the color development occurs by combining with the bovine serum albumin conjugate layer (2), the color development of the test line appears stronger than that of the control line. In this way, by observing the relative intensity of color development between the test line and the control line, pregnancy/non-pregnancy of the sample can be easily determined. That is, steroid hormones such as progesterone (20) present in the blood are combined with the anti-progesterone immunoglobulin gold-labeled conjugate (10), and when they gradually reach the position where progesterone is fixed (2), progesterone in the test sample (20) and progesterone-bovine serum albumin immobilized on the thin film combine with the anti-progesterone immunoglobulin gold-gold label conjugate (10) in a competitive assay, inversely proportional to the content of progesterone contained in the analysis sample. The degree of bright red coloration of the test line is different. Therefore, it is possible to determine whether the sample is pregnant by visually grasping the degree of color development of the progesterone-bovine serum albumin conjugate layer 2 . The anti-progesterone immunoglobulin G-gold labeled conjugate that passed without binding to the progesterone-bovine serum albumin conjugate layer (2) was anti-immune to the control line immobilized with anti-immunoglobulin (sheep-derived anti-mouse immunoglobulin G: 3). It is detected by binding to globulin paper (sheep-derived anti-mouse immunoglobulin paper: 3) and developing color. The amount of colloidal gold conjugate detected at the anti-immunoglobulin (anti-IgG) immobilized site is directly proportional to the amount of progesterone (4) in the assay sample. Therefore, the user (animal stock) is concerned with the bright red band color or The degree of coloration is relatively compared to determine whether the livestock is pregnant. If the degree of coloration of the test line is lower than that of the control line, it is judged to be pregnancy.

Using the pregnancy diagnosis kit according to the present invention as described above, the pregnancy status of Korean cattle was determined on the 19th, 20th and 21st days after artificial insemination and is shown in Table 1 below.

reproductive status
Days after artificial insemination pregnancy not pregnant Positive reaction/number of pregnant cows (%) Negative reaction/number of non-pregnant cattle (%) 19th 17/20 (88.2) - 20 days 184/195 (94.2) 82/82 (96.2) 21st 73/75 (97.3) 32/33 (97.1) Sum 275/290 (93.2) 114/118 (96.7)

As can be seen from Table 1, the pregnancy diagnosis kit according to the present invention exhibited a diagnostic accuracy of 93.2% in the case of a pregnancy diagnostic test and a diagnostic accuracy of 96.7% in the case of a non-pregnancy diagnostic test. Therefore, the pregnancy diagnosis kit of the present invention can diagnose pregnancy/non-pregnancy with a high degree of accuracy. As described above, the pregnancy diagnosis kit of the present invention can discriminate with high accuracy an animal in which fertilization has failed after artificial insemination, and thus has relatively superior economic feasibility compared to diagnostic kits having a high non-pregnancy diagnosis rate.

1: thin paper
2: progesterone-bovine serum albumin conjugate layer
3: anti-immunoglobulin
6: sample absorbent paper
10: anti-progesterone immunoglobulin gold-gold labeled conjugate
20: progesterone

Claims (18)

Analysis in which one end of the perforated thin-film paper is coated with a progesterone-BSA conjugate as a test line and the other end is coated with anti-immunoglobulin (anti-IgG) as a control line. strip; and
An animal pregnancy test kit consisting of anti-progesterone immunoglobulin gold-labeled conjugate (anti-progesterone IgG-gold conjugate).
The pregnancy diagnosis kit according to claim 1, wherein the thin-film paper is formed with a plurality of micropores having a diameter of 5 to 12 μm.
The pregnancy diagnosis kit according to claim 1, wherein the thin paper is made of a nitrocellulose membrane.
The pregnancy diagnosis kit according to claim 1, wherein the interval between the test line and the control line of the assay strip is 8 to 12 mm.
The pregnancy diagnosis according to claim 1, wherein the anti-immunoglobulin (anti-IgG) of the control line is anti-immunoglobulin (anti-IgG) derived from an animal selected from the group consisting of mouse, goat, and sheep. kit.
The method of claim 1, wherein the anti-immunoglobulin (anti-IgG) of the control line is from the same species as the immunoglobulin (IgG) of the anti-progesterone immunoglobulin gold-labeled conjugate (anti-progesterone IgG-gold conjugate) A pregnancy diagnosis kit, characterized in that obtained by immunizing the derived immunoglobulin.
The pregnancy test kit according to claim 1, wherein the anti-progesterone immunoglobulin Z-gold labeled conjugate is mixed with a stabilizer and lyophilized.
The pregnancy diagnosis kit according to claim 7, wherein the stabilizer is at least one selected from the group consisting of bovine serum albumin and sucrose.
The pregnancy diagnosis kit according to claim 1, wherein the anti-progesterone immunoglobulin Z-gold labeled conjugate is dissolved in a phosphate buffer containing a nonionic surfactant.
10. The pregnancy test kit according to claim 9, wherein the nonionic surfactant is at least one nonionic surfactant selected from the group consisting of BSA, Tween 20 and Tween 40.
The pregnancy diagnosis kit according to claim 1, wherein the pregnancy diagnosis kit diagnoses the pregnancy of an animal 19 to 21 days after fertilization.
mixing an anti-progesterone immunoglobulin gold-labeled conjugate with a sample for analysis;
Test line of an assay strip coated with a progesterone-BSA conjugate as a test line on one end of a thin paper and coated with anti-IgG as a control line on the other end. immersing the coated end in the mixed solution;
A method for diagnosing pregnancy in an animal, comprising comparing intensity of color development between the test line and the control line to determine whether the animal is pregnant.
The method for diagnosing pregnancy according to claim 12, wherein the sample for analysis is one or more samples selected from the group consisting of serum, plasma and milk separated from the subject.
The method of claim 12, wherein the progesterone in the sample for analysis and the progesterone of the progesterone-bovine serum albumin conjugate react competitively with the anti-progesterone immunoglobulin gold-gold conjugate. A method for diagnosing pregnancy in animals.
The method for diagnosing pregnancy according to claim 12, wherein the animal is one or more animals selected from the group consisting of Korean cattle, dairy cows, and cattle.
The method for diagnosing pregnancy according to claim 12, wherein the color of the control line appears stronger than that of the test line when the concentration of progesterone in the mixed solution is 5 ng/ml or more in the step of determining whether the animal is pregnant.
The method for diagnosing pregnancy according to claim 12, wherein the color of the test line is stronger than that of the control line when the concentration of progesterone in the mixed solution is 1 ng/ml or less in the step of determining whether the animal is pregnant.
[Claim 13] The method for diagnosing pregnancy according to claim 12, wherein the step of determining whether the animal is pregnant is determined by visually comparing intensity of color development.

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