KR100574559B1 - Kit for detecting canine parvovirus antibody using immunochromatography and manufacturing method thereof - Google Patents

Abstract

The present invention relates to an antibody diagnostic kit that can quantitatively detect canine parvovirus antibodies using the principle of rapid immunochromatography, and a method for preparing the same. More specifically, a monoclonal specific for canine parvovirus VP2 protein The invention relates to a diagnostic kit and a method for manufacturing the diagnostic kit using the conjugated gold conjugated antibody and the dog parvovirus using a rapid immunochromatography method, and to rapidly measure the dog parvovirus antibody titer in the blood of a dog, which is a test material. to be.

To this end, the present invention is a process for preparing canine parvovirus and monoclonal antibody, adsorbing a goat anti-dog IgG antibody to the nitrocellulose membrane, combining the monoclonal antibody and gold conjugate and drying on a gold pad, purified The step of drying the canine parvovirus on the sample pad, the step of drying the absorbent pad is absorbed by the combination, characterized in that the step of assembling to the inspection device after the step.

Canine parvovirus, rapid immunochromatography, antibody diagnostic kit

Description

Kit for detecting canine parvovirus antibody using immunochromatography and manufacturing method according to rapid immunochromatography method

1 is a photographic cross-sectional view taken a diagnostic kit according to the present invention

2 is a structural diagram of a diagnostic kit used in the present invention

Figure 3 is a dog babovirus monoclonal antibody western blot photograph used in the present invention

Figure 4 is an example of the reaction result after the application of the sample of the diagnostic kit according to the present invention

<Description of the code | symbol about the principal part of drawing>

1: sample pad 2: gold pad

3: control line 1 4: inspection line

5: control line 2 6: moisture absorption pad

7: Device

Canine parvovirus (CPV), one of the most important diseases in dogs in Korea, is a virus belonging to Parvoviridae. It is 18 ~ 22nm in size, has single stranded DNA, has a cubic symmetry capsid, and has 1.2 ~ 1.8 without envelope. It is a small x10 ⁶ dalton virus that causes the VP2 protein in its viral structure to form antibodies.

CPV is commonly separated from healthy dog feces and is divided into non-pathogenic CPV-1 and CPV-2, which causes severe enteritis and cardiac disease and damages the dog. CPV-2 is mainly caused by hemorrhagic enteritis and myocarditis. Clinically important acute infectious diseases such as hemorrhagic enteritis and myocarditis have high mortality rate, vomiting, lack of appetite, bloody stool, fever, dehydration and leukopenia. As the causative agent of, it has been distributed worldwide since it was first reported and separated by Binn et al. In 1970.

In order to prevent canine parvovirus, the dog parvovirus vaccine is usually given 3 to 4 times from 6 weeks of age. However, vaccination varies from 0% to 100% depending on the titer level of maternal antibody, so when the maternal antibody titer is less than 1:80 as the hemagglutination inhibition titer at the time of vaccination, Inoculation may have the best vaccine effect.

Diagnosis of canine parvovirus infection is possible by combining hemagglutination and hemagglutination inhibition of fecal samples, and for confirmation, indirect fluorescence antibody detection, virus isolation, electron microscopy, and confirmation of serum conversion. .

Recently, dog parvovirus antigen detection using immunochromatography and enzyme immunoassay for antibody measurement have been developed, and the pre- and post-vaccination antibodies of dog parvovirus infection or vaccination have been widely used in animal hospitals.

Serum diagnostic methods to date include enzyme immunoassay and hemagglutination inhibition. However, these methods require specific equipment and fresh porcine erythrocytes, making them difficult to carry out in general veterinary clinic sites as well as inconvenient testing time of at least 4-5 hours to 1 day. These shortcomings do not result in an immediate test results for dogs visited to the animal hospital, there is a problem that the owner should come back.

In order to solve the above problems, the present invention provides an antibody-antibody-antigen indirect sandwich method using purified canine parvovirus antigen and goat anti-canine IgG antibody. We developed a diagnostic kit designed to detect and detect the presence of canine parvovirus antibodies and the degree of antibody titer quickly and easily within 20 minutes after the test.

The reaction principle of the present invention is as follows.

When the sample solution containing dog parvovirus antibody (1 μl of blood and 210 μl of diluent) was placed in the sample application hole, the dog parvovirus antibody in the sample binds to the dog parvovirus antigen on the sample pad, and then the mouse dog parvo with gold particles Reacts with viral monoclonal antibodies to form a complex. The binding moves on the membrane by capillary action and again reacts with goat anti-dog IgG antibodies adsorbed on the membrane to form a complex of antigen-antibody-membrane antibodies by the indirect sandwich principle. A purple band (inspection line / T) forms in place. Pig IgG gold particles that did not react on the test line reacted at control sites where goat anti-pig IgG antibodies were adsorbed (control 1 / C1 and control 2 / C2) to show purple bands of different color development.

That is, the sample solution to be tested is placed in an elliptical sample application hole and observed for 20 minutes after the purple band appears at the C1, T, and C2 positions. In all cases, purple lines of different color development should appear at C1 and C2 positions, and the color development of purple lines at T position is compared with C1 and C2 to determine the presence or absence of antibody and antibody titer.

In order to achieve the above object, the present invention provides a method for preparing canine parvovirus and monoclonal antibody, adsorbing goat anti-dog IgG antibody to nitrocellulose membrane, conjugating monoclonal antibody and gold particles and drying them on a gold pad. And drying the purified canine parvovirus on the sample pad, drying the moisture absorbent pad where the combinations are absorbed, and assembling the test dog parvovirus into the test device after the step.

This immunochromatography is characterized by the process of sample dilution, washing, and reaction of enzyme-conjugate and substrate, which can be found in the existing multi-step immunoassay. It is convenient to test all in one step. It also has the advantages of ease and economy of determining the test results without using specific equipment, and the speed of reading the test results.

In the specific method, dog parvovirus was first grown in CRFK cells, and the virus was purified and purified by Affinitiy chromatography. This isolated canine parvovirus produced a monoclonal antibody against canine parvovirus. Goat anti-dog IgG antibodies were purified by IgG in dog serum with Protein G, immunized with goats four times, and then goat blood was collected and IgG was purified again to prepare goat anti-dog IgG antibodies.

To diagnose by immunochromatography, goat anti-dog IgG antibodies were adsorbed to nitro cellulose membranes at fixed positions, and dog parvovirus monoclonal antibodies were conjugated to gold particles and dried on gold conjugate pads.

Sample pads to which the sample is applied were prepared by soaking and drying the purified canine parvovirus.

A test strip was prepared by overlapping the dried gold conjugate pad and the sample applying pad and covering the nitrocellulose membrane with a moisture absorbent pad at an opposite position.

1 is prepared by inserting a diagnostic strip prepared according to the diagnostic kit manufacturing method of the present invention in a plastic device in which a sample application hole and a result display window are displayed.

Membranes usable in the manufacture of diagnostic strips according to the present invention can utilize those used as materials for conventional diagnostic strips, for example, nitro cellulose, cellulose, cellulose acetate, polyethylene, etc. Synthetic polymers can be used.

Goat anti-pig IgG was adsorbed at the control line, so that the gold particles adsorbed with pig IgG were always reacted regardless of the presence of canine parvovirus antibody in the sample to show a purple band. The unreacted content penetrates into the absorbent pad so that the membrane of the inspection window appears clean white, making the formed band easy to read. In other words, if a certain amount of canine parvovirus antibody is present in the sample, the color will be quantitatively developed according to the amount.

Hereinafter, a diagnostic apparatus and a manufacturing method according to the present invention will be described in more detail with reference to the following examples.

Example 1 Canine Parvovirus Purification and Monoclonal Antibody Preparation

1) Cultivation and Purification of Canine Parvovirus

Protovirus for canine parvovirus purification was purchased from ATCC (VR-953, C-780916) and was inoculated with virus when about 80% of CRFK (Crandell Feline Kidney Cell) sheets were formed.

Purification of the virus was performed after 48-72 hours of virus inoculation, when cell denaturation reached 100%. After the cell suspension was recovered, the cells were centrifuged at 3,000 rpm and 10 min through three freeze thaws. After centrifugation, the virus was purified by inactivation at the final concentration of formalin of 0.2% at room temperature for 24 hours, and the supernatant showing more than 1,024 fold HAU using porcine erythrocytes. For purification, the supernatant was added with 4% PEG-8000, NaCl 5M, stirred at 4 ° C., and resuspended in 50 mM Tris-Cl (pH 8.0). The precipitate was extracted with chloroform, centrifuged at 100,000 x g for 2 hours, suspended in PBS and sucrose gradient ultra-centrifugation (SGUC) to obtain virus fractions. The antigen thus purified was used for preparation of mouse canine parvovirus monoclonal antibody and sample pad.

2) Mouse Canine Parvovirus Monoclonal Antibody Preparation

The purified canine parvovirus antigen was used as an immunogen to immunize Balb / c mice. Afterwards, splenocytes were isolated, conjugated with myeloma, and hybrid cell lines secreting monoclonal antibodies against canine parvovirus antigen were selected by ELISA, and several mononuclear cell groups were established by limiting dilution. These cell lines were mass cultured and inoculated to Balb / c mice intraperitoneally to obtain ascites containing a large amount of antibody, and IgG antibody was purified using protein A / G gel. This purified antibody was confirmed by western blot that the monoclonal antibody against 65 kDa of the gabbovirus VP2 (Fig. 3). Purified antibody was diluted with PBS at a concentration of 1 mg / ml and used for gold conjugation.

Example 2 Preparation of Goat Anti-Canine IgG Antibody for Test Ship

Purified dog IgG was purchased, dissolved in physiological saline, and prepared at 100 ㎍ for each inoculation, followed by emulsion with Freund's Adjuvant, complete 0.5 ml. The inoculum thus prepared was inoculated subcutaneously over 30 kg of goats. Six times a month was inoculated in the same manner, but from the second inoculation was used incomplete adjuvant.

Test blood was collected from the jugular vein of an immunized goat. Serum was isolated and titer was measured by ELISA. The titer was measured by adsorbing dog IgG at a concentration of 1 ㎍ / ㎖ on ELISA plate and diluting the antiserum with 1% BSA solution to 1,000, 10,000, 100,000, 1,000,000 times. Secondary reaction is carried out using the anti-goat IgG-peroxidase purchased and developed with TMB. Whole blood was collected when the absorbance, which was about three times more than the absorbance of normal goat serum, was cut off and showed a titer of 1 million times or more.

Serum was isolated by centrifugation after whole blood collection, and dog IgG was adsorbed on sepharose 4B gel and passed. In antisera, goat anti-canine IgG antibodies responded to dog IgG and unreacted proteins were washed with PBS.

Specific antibodies were eluted with 0.2 M glycine (pH 2.0) solution and the eluate was dialyzed with PBS. Protein concentration of the dialyzed antibody solution was measured by BCA method and stored in the refrigerator until use.

Example 3 Preparation of Goat Anti-Swine IgG Antibody for Control

Purified porcine IgG was purchased, dissolved in physiological saline, and prepared at 100 ㎍ for each inoculation, followed by mixing with Freund's Adjuvant, complete 0.5 ml and emulsion. The inoculum thus prepared was inoculated subcutaneously over 30 kg of goats. Six times a month was inoculated in the same manner, but from the second inoculation was used incomplete adjuvant.

Test blood was collected from the jugular vein of an immunized goat. Serum was isolated and titer was measured by ELISA. The titer was measured by adsorbing porcine IgG at a concentration of 1 ㎍ / ㎖ on ELISA plate and diluted antiserum to 1,000, 10,000, 100,000, 1,000,000 times with 1% BSA solution. Secondary reaction is carried out using the anti-goat IgG-peroxidase purchased and developed with TMB. When the absorbance at least three times greater than the absorbance of normal goat serum was cut off, a titer of more than one million times resulted in whole blood collection.

Serum was isolated by centrifugation after whole blood collection, and dog IgG was adsorbed on sepharose 4B gel and passed. In antisera, goat anti-canine IgG antibodies responded to dog IgG and unreacted proteins were washed with PBS.

Specific antibodies were eluted with 0.2 M glycine (pH 2.0) solution and the eluate was dialyzed with PBS. Protein concentration of the dialyzed antibody solution was measured by BCA method and stored in the refrigerator until use.

Example 4 Preparation of Mouse Canine Parvovirus Monoclonal Antibody-Gold Conjugate

Gold chloride was dissolved in distilled water at 0.01% and heated with 0.1% sodium citrate solution. It was used to react with the gold conjugate while heating for about 10 minutes and then cooled and refrigerated. Mouse canine parvovirus monoclonal antibody was added to the gold solution at a concentration of 1 mg / ml and reacted for 10 minutes. After stabilizing the gold particles were centrifuged at 10,000g for 30 minutes to form a precipitate, dissolved in PBS again and filtered with a 0.45㎛ filter and diluted to a absorbance of 10 at 540nm. This gold solution was mixed with Tween 20 and transferred to a goat anti-mouse immunoglobulin-adsorbed membrane to confirm reactivity.

Example 5 Pig IgG-Gold Conjugate Preparation Method

Gold chloride was dissolved in distilled water at 0.01% and heated with 0.1% sodium citrate solution. It was used to react with the gold conjugate while heating for about 10 minutes and then cooled and refrigerated. Porcine IgG antibody was added to the gold solution at a concentration of 1 mg / ml and allowed to react for 10 minutes. After stabilizing the gold particles were centrifuged at 10,000g for 30 minutes to form a precipitate, dissolved in PBS again and filtered with a 0.45㎛ filter and diluted to a absorbance of 10 at 540nm. This gold solution was mixed with Tween 20 and transferred to a goat anti-mouse immunoglobulin-adsorbed membrane to confirm reactivity.

Example 6 Strip Production Method

1) Membrane Coating Process

Goat anti-dog IgG (1.0 mg / mL) antibody at a specific location on the nitrocellulose membrane, ie, test T, goat anti-pig IgG antibody at control lines C1 (0.1 mg / mL) and C2 (1.0 mg / mL). Each coat.

2) Gold conjugate pad (gold pad) manufacture

Mouse dog parvovirus monoclonal antibody-gold conjugates and porcine IgG-gold conjugates were soaked in polyester or glass fibers and dried to prepare gold pads.

3) sample pad manufacturer

Purified canine parvovirus antigen was soaked in a sample pad to 2 ¹² HAU and dried to prepare a sample pad.

4) Hygroscopic pad manufacturer

A moisture absorbing pad is prepared by drying the reaction solution so that the reaction solution can be absorbed well.

5) strip assembly

A gold pad, a sample pad, and a moisture pad are attached to the prepared membrane to prepare a strip.

Example 7 Test Principle of Canine Parvovirus Antibody Diagnostic Reagent by Rapid Immunochromatography

When the sample solution (serum or blood) containing the canine parvovirus antibody is put into the sample application hole (S), the canine parvovirus antibody in the sample binds to the canine parvovirus antigen on the sample pad, and then the mouse canine parvovirus with gold particles Reacts with monoclonal antibodies to form a complex. The binding moves on the membrane by capillary action and again reacts with goat anti-dog IgG antibodies adsorbed on the membrane to form a complex of antigen-antibody-membrane antibodies by the indirect sandwich principle, in which case the color of the gold particles A purple band (inspection line / T) forms in place. Pig IgG gold particles that did not react on the test line reacted at control sites where goat anti-pig IgG antibodies were adsorbed (control 1 / C1 and control 2 / C2) to show purple bands of different color development.

That is, the sample solution to be tested is placed in an elliptical sample hole and observed for 20 minutes after the purple band appears at the C1, T, and C2 positions. In all cases, purple lines of different color development should appear at C1 and C2 positions, and the color development of purple lines at T position is compared with C1 and C2 to determine the presence or absence of antibody and antibody titer.

Example 8 Diagnostic Efficacy Test of Diagnostic Kit According to the Present Invention

1) Correlation with Hemagglutination Inhibition

The titer correlation with the present invention was investigated in dog sera whose dog parvovirus antibodies were known for hemagglutination inhibition, which is a standard test for measurement.

Hemagglutination Inhibition Titer / HI titer was performed according to the standard serological test of livestock disease from National Veterinary Research and Quarantine Service. That is, in order to remove the nonspecific reaction of the serum first, the serum was diluted 1: 4 with Sorenson buffer (pH6.8), and 50% Kaolin was mixed with the diluted serum in the same amount and left at room temperature for 1 hour. After centrifugation for 10 minutes at 1,500rpm, 1/10 dose of 50% porcine red blood cell solution was added to the supernatant, and then left to stand at room temperature for 1 hour. After standing, the supernatant was disclosed in HI test by centrifugation at 1500 rpm for 10 minutes. HI reaction was carried out as follows. That is, 25 μl of Sorenson buffer (pH6.8) was added from wells 2 to 12 of 96 well plates of U buttom, and 50 μl of serum from which the nonspecific reaction was removed was added to each well. Dilute the dilution steps in 25ul increments from wells 1 to 12 wells and discard the last 25µl. In each well of the step-diluted serum, 25 µl of 8HAU canine parvovirus was added to the whole well to neutralize the virus and serum for 1 hour at room temperature. Then, 50 μl of 1% chicken erythrocyte suspension was added and shaken well, followed by overnight at 4 ° C. at room temperature. Hemagglutination inhibition titer was determined as the highest dilution factor that does not aggregate erythrocytes.

In the present invention, the sample dilution solution (210 μl) and serum 1 loop (1 μl) were mixed well in the sample application hole, and after 20 minutes, negative, 1+, 2+, 3, depending on the development of the test line (T) It was determined as +.

As a result, if the HI titer is less than 1:10, it is negative, if it is more than 1:10 and less than 1:80, it is 1+, from 1:80 or more is less than 1: 640 to 2+, and if it is more than 1: 640, it is 3+. When the HI antibody titer was high, the intensity of the test line was also increased proportionally.

2) sensitivity, specificity test

In order to test the sensitivity and specificity of the present invention, 286 whole blood, plasma, and serum samples were supplied from the animal hospital affiliated with Cheju National University College of Agriculture and the animal hospital in Seoul, and the blood clotting inhibitory activity was compared with the present invention. As shown in Table 1, the sensitivity was 100% (274 / 274x100) and the specificity was 100% (12 / 12x100).

 As described above, the present invention, as described above, can be used for the rapid diagnosis of canine parvovirus antibody according to the present invention using the blood, serum, and plasma of the dog in 20 minutes without any special test equipment in the field of animal hospital It is a useful test method that can be diagnosed simply and quickly. Thus, dog parvovirus antibodies may be helpful for writing vaccine programs or diagnosing dog parvoviruses in veterinary clinic sites based on measurements.

Claims (4)

On a strip coated with goat anti-dog IgG antibody-coated membrane, a gold pad conjugated with canine parvovirus-specific monoclonal antibody and gold, a sample pad impregnated with canine parvovirus antigen, and a absorbent pad for absorbing reaction solution A canine parvovirus antibody diagnostic kit by rapid immunochromatography, characterized in that the canine parvovirus antibody titer in dog blood is measured. The canine parvovirus antibody diagnostic kit according to claim 1, wherein the display window of the diagnostic kit is configured in the order of the control line 1, the test line, and the control line 2. The method of claim 2, wherein the control line 1 of the diagnostic kit indicates that the titer of the dog parvovirus hemagglutination inhibitory antibody is 1:80, and the control line 2 causes the titer of the dog parvovirus hemagglutination inhibitory antibody to be colored with the same intensity as 1: 640. Canine parvovirus antibody diagnostic kit by rapid immunochromatography. A membrane manufacturing process of coating a goat anti-dog IgG antibody at a specific position (test line) of the membrane; A gold pad manufacturing step of moistening a mouse dog parvovirus monoclonal antibody-gold conjugate and a swine IgG-gold conjugate on a pad; A sample pad manufacturing step of impregnating and drying the purified canine parvovirus antigen to the sample pad at 2 ¹² HAU; A moisture pad manufacturing step of drying the reaction solution to absorb moisture; A method for producing a dog parvovirus antibody diagnostic kit by rapid immunochromatography, which is a step of attaching a gold pad, a sample pad, and a hygroscopic pad to the membrane to prepare a stream.

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