KR100261050B1 - Swine Ozeski's Disease Antibody Testing Device Using Immunochromatography - Google Patents

Abstract

PURPOSE: Provided is a device for detecting antibody of Aujeszky's disease. And a method for diagnosing Aujeszky virus more rapidly and correctly using the same is also provided. CONSTITUTION: The device for detecting antibody of Aujeszky's disease is composed of; i) a nitrocellulose membrane (5), absorption pad (3), supporting plate (6), and soaking section (4). The method for detecting antibodies of Aujeszky virus is comprised of the following steps of: i) spreading antigens (1) of Aujeszky virus and anti-swine IgG (2) evenly on the nitrocellulose membrane (5); and ii) reacting the antigens (1) and IgG (2) with antibodies of Aujeszky virus in the soaking section (4), which shows different colors depending on the existence of antibodies.

Description

Swine Ozeski's Disease Antibody Testing Device Using Immunochromatography

The present invention relates to a method for detecting antibodies to porcine ozesky virus using a test container in which an antigen is fixed for detecting specific antibodies to porcine ozesky virus in a short time using an antigen antibody reaction.

Conventional methods for detecting antibodies against porcine Ozeskiviruses include virus neutralization test (VN test), fluorescent antibody-labeled specific antibodies, and enzyme-linked immunosorbent assays. ELISA) and latex agglomeration reactions are used, but they require specialized manpower and experimental equipment, and the detection time is long, which makes them all unsuitable for outdoor use. Neutralization test can indirectly measure the amount of antibody, but the pig blood is collected and collected only by primitive separation of serum components, and then boiled at 56 ° C for at least 30 minutes, and the test takes 3 to 5 days. in need. Fluorescent antibody method also needs to purify specific antibodies against Ozeskiviruses and label them with fluorescent materials, and they cannot be used in outdoor farms because they require special equipment such as fluorescence microscope and dark room. In addition, the antibody detection method using Eliza and latex aggregation reaction has the advantage of processing a large amount of serum samples, but these methods also take 4 to 5 hours and are used in pig farms because they require expert personnel and equipment to read the results. Not suitable below.

Therefore, in the present invention, paying attention to the existing uncomfortable antibody detection method, the blood is collected in the absorbent from pigs, immersed in an appropriate amount of buffer, and the nitrocellulose membrane in which the purified purified Ozeskivirus protein is fixed in a linear form is in a strip form. When cleaved and immersed in the buffer in which the blood absorbent paper is immersed, the antibody contained in the blood moves through the membrane to the place where the Ozeskivirus antigen is fixed. When anti-swine immersed in anti-swine IgG-labeled gold particle buffer, the color reaction occurs linearly on the membrane to which the antigen is fixed according to the presence or absence of antibody against Ozeskivirus in suspension buffer. The method for detecting an antibody against Ozeskivirus was completed.

As in the case of swine Ozeski's disease, there is an urgent need for a test method that can carry out antibody tests on a farm as quickly as possible. Therefore, the immunochromatography method can be used to quickly determine whether or not the pig has an antibody against Ozeski's disease. This has been required. There is a need for a faster and more accurate viral antibody test than the virus neutralization test, fluorescent antibody method, and ELISA and latex agglutination method using enzyme-detecting antibodies. Hereinafter, the present invention will be described in detail.

Figure 1 is a photograph of the denaturation effect of domestically isolated swine Ozeski virus mass production by immunocytochemistry (immunocytochemistry) method.

2 is a schematic diagram of the purification of Ozeskivirus antigen used in the present invention.

3 is a schematic diagram of a diagnostic method used in the present invention.

4 is a schematic diagram of blood absorbent paper.

5 is a schematic diagram of a diagnostic kit used in the present invention.

FIG. 6 is a schematic diagram showing the positive and negative response and the result photograph when antibody to Ozeskivirus is present in porcine blood.

* Explanation of symbols for main parts of the drawings

1: Swine Ozeski virus antigens 2: Anti-swine anti-globulin eye support antibody

3: absorption pad 4: immersion part

5 Nitorocellulose Film 6 Support Plate

7: Gold or latex conjugate solution 8: Transparent polystyrene tube

Example 1

[Pig Blood Collection and Processing]

The test blood used in the present invention can be collected directly from pigs and passed through a filter for removing red blood cells, or blood collected at the time of hog vein, tail vein, or slaughter to the cellulose absorbent paper as shown in FIG. Absorb. This is again used by eluting serum components by dipping for 10 minutes in a transparent tube containing 500 μl of Tris buffer solution (Tris buffered saline: TBS, Tris 50mM, NaCl 150mM pH 7.2).

Example 2

[Production of Porcine Ozuki Virus Antigen for Diagnosis]

Passage was performed in porcine kidney (PK15) isolated from domestically isolated Ozeski virus parasites. Cell culture medium was 5% fetal calf serum (FCS, Hybriserum, Austria) in alpha minimal essential medium ), Non-essential amino acid (BRL) and gentamicin (50 µg / ml) were added to the cell, and the cell denaturation effect was more than 95% after incubation at 37 ° C for 3 days after 0.1 moi inoculation. When the culture supernatant was centrifuged at 10000 × g for 20 minutes, the supernatant was collected in a sterile container and stored in a freezer at -70 ° C to carry out virus titer test to confirm that it was 10 ⁸⁰ TCID ₅₀ / ml or more. It was dialyzed overnight in neutral tris buffer solution, and 5% of trehalose sugar was completely dissolved and then subdivided and stored at -70 ° C and used as an antigen (see FIGS. 2 and 3).

Example 3

[Preparation of Nitrocellulose Membrane Strips with Ozeski Virus Antigens Fixed]

The solution containing the dissolved Ozeskivirus antigen protein prepared in Example 2 was placed in an injector (BioDot. USA) and sprayed onto a nitrocellulose membrane under the same conditions as in Table 1. The injection position of the Ozeskivirus antigen is about 15 mm from the nitrocellulose membrane submerged with the blood buffer solution, and the anti-pigment reverse globulin eye support antibody used as a control antigen is 10 mm away from the position where the Ozeski virus antigen is injected. Spray on location. The antigen-injected nitrocellulose membrane was dried for 39 minutes at 37 ° C and immediately adhered to an adhesive-coated polypropylene plate membrane. They were attached together and cut as shown in FIG. 5 to complete the Ozeskivirus antibody test strip.

Example 4

[Production of conjugated adsorbed reverse globulin eye support]

ㅇ Gold conjugated with anti-swine reverse globulin eye support

Anti-swine IgG (Pierce Co. cat.no. 31231) was dialyzed in 2 mM Borax buffer (Borax buffer (sigma, pH 9.0)) at a concentration of 2 µg / µl and finally 0.1 µg. Dilute to / μl. On the other hand, colloidal gold (Arista Biological Inc) having a diameter of 40 nm is diluted to a concentration of 0.957 at an absorbance of 530 nm and the pH is adjusted to 100 mM K ₂ CO ₃ to 9.0. When added to the prepared gold solution, quickly add reverse globulin eye support solution and stir for 5 minutes. Add 1/10 volume 10% bovine serum albumin (BSA, pH 0.9) and stir for 10 minutes. Gold particles to which the antibody was adsorbed were harvested by centrifugation at 5000 rpm for 50 minutes, which were then suspended in 1/10 of the initial volume in a Tris buffer solution (50 mM Tris, 150 mM NaCl, pH 8.2) containing 1% BSA.

ㅇ Anti-swine IgG: Lattes conjugated with anti-swine IgG (Jackson Co. cat.no.)

Anti-swine IgG (Pierce Co. cat.no. 31231) is dialyzed in distilled water and lyophilized. Finally, 30 ml was prepared by diluting with distilled water to 2% w / v, followed by 800 ml of antibody adsorption buffer (0.15% glycine, 0.2% NaCl, pH 9.2) and blocking buffer (2% BSA, 0.15% glycine, 0.2%). NaCl, pH 9.2) 300 ml and 100 ml of a 10% latex suspension with a diameter of 0.2-0.3 μm were added and mixed thoroughly for 15-30 minutes. The reaction was carried out at room temperature for 2 hours and then precipitated by centrifugation at 10000rpm for 50 minutes, and again, 0.5% in a solution containing a preservative buffer (1% BSA, 1% NaCl, 0.75% glycine, 0.01% thimersal, pH 8.2). Very rich.

Anti-pigment reverse globulin eye support gold conjugate or anti-pigment reverse globulin eye support latex conjugate produced as described above was stored in 4 ℃. When used, shake well and then dispensed 300-500 μl into a transparent cylindrical tube as shown in FIG.

Example 5

[Tristowin buffer (Tris 50mM, NaCl 150mM, 0.1% Tween 20, pH 7.2: TBST)]

[Detection of Swine Ozeski Virus Antibodies in Porcine Blood]

A blood sample treated with nitrocellulose strips fixed with porcine Ozeski antigen prepared by the method of Example 3 was treated with Tristwin buffer (Tris 50 mM, NaCl 150 mM, 0.1% Tween 20, pH 7.2: TBST). The submerged portion of Ozeskivirus virus antigen is immersed in a transparent cylindrical tube of) and then reacted for 10-15 minutes at room temperature. At this time, shake the container containing the gold conjugate or latex conjugate to which the reverse globulin eye support is adsorbed, and then dispense 300 to 500 μl into a transparent cylinder tube. After the antibody reaction is completed, the string is transferred to a tube tube dispensed with gold conjugate, immersed and reacted for 10 to 15 minutes, and then colored in a line in the nitrocellulose stream to which the antigen is fixed. If the color appears at the same time linearly, it is determined to be positive.If the color does not come out linearly on the nitrocellulose strip to which the antigen is fixed, the color becomes linear if only the area where the reverse globulin is fixed becomes negative. 6 also obtained the same result.

Swine Ozeski disease antibody detection using immunochromatography is a method of detecting a specific antibody in a short time by immersing a nitrocellulose membrane immobilized with an Ozeskivirus antigen in a buffer containing the antibody using gold or latex particles sensitized to the antibody. A method for preparing an antibody test kit useful for rapidly detecting and diagnosing Ozeski's disease from pig blood, and color reactions at the sites where the Ozeskivirus antigen and anti-pigment reverse globulin were fixed, can be used to determine the presence of antibodies.

Claims (3)

One end of the nitrocellulose membrane (5) and the absorption pad (3) are attached to the support plate (6), and the pig Ozeskivirus antigen (1) and the anti-pig immunoglobulin eye support antibody (2) on the nitrocellulose membrane (5) at regular intervals. When the pig Ozesky virus antibody immersed in the immersion unit 4 is fixed by spraying to the porcine Ozesky virus antigen (1) and the anti-swine immunoglobulin eye support antibody (2), the color reaction is performed to determine the presence of antibodies. Pig Ozeski virus antibody testing apparatus using immunochromatography. Swine Ozeskivirus antigen and anti-pigment reverse globulin are sprayed and fixed to nitrocellulose membranes at a predetermined interval, and then reacted first in a buffer tube containing swine blood, and anti-pigment anti-globulin eye support gold or latex conjugate buffer Piglet Ozeski virus antibody testing apparatus using immunochromatography to determine the presence of antibodies in a short time by the color reaction appearing at the position where the porcine Ozeski virus antigen and anti-pigment reverse globulin are fixed after the second reaction. According to claim 2, Porcine Ozuki virus antigen is characterized by the production of porcine Ozuki virus virus and immunochromatase characterized in that it contains Tris buffered solution (Tris buffered saline: TBS, Tris 50mM, NaCl 150mM pH 7.2) Pigment Ozeski virus corpus luteum test using the graphigraphy method.