CN102313803A - Goat pox indirect hemagglutination diagnosis reagent as well as preparation method and detecting method thereof - Google Patents

Abstract

The invention discloses a goat pox forward indirect hemagglutination diagnostic reagent and a preparation method and a detection method thereof. The goat pox virus antigen-sensitized red blood cell content in the hemagglutination diagnostic reagent is 0.6-1.2%, and the purified goat pox virus antigen content is Compared with the existing commonly used agar diffusion test, the present invention prepares goat pox positive indirect hemagglutination diagnostic reagent by purifying goat pox virus antigen sensitized aldehydated sheep red blood cells, which can be fast, accurate and simple The detection of goat pox virus antibody can be judged within 50 minutes, and can detect antibodies with lower titer, which has good specificity, sensitivity and repeatability, and can be widely used in veterinary clinical goat pox The rapid and large-scale detection of virus antibodies provides a basis for disease prevention, diagnosis, and prevention and control.

Description

Goat pox positive indirect hemagglutination diagnostic reagent and its preparation method and detection method

technical field

The invention relates to the technical field of positive hemagglutination diagnostic reagents, in particular to a goat pox forward indirect hemagglutination diagnostic reagent and a preparation method and a detection method thereof.

Background technique

Goat pox is an endemic highly contagious infectious disease caused by goat poxvirus (GTPV), which can occur all year round and is common in spring and autumn, regardless of gender, age, or species. The World Organization for Animal Health (OIE) listed goat pox as a major infectious disease of category A, and in 1999, the Ministry of Agriculture of the People's Republic of China announced goat pox as a national first-class animal disease. In recent years, more than 20 provinces and cities in my country have successively reported the occurrence of goat pox epidemic, which has caused great economic losses and posed a serious threat to the booming goat breeding industry in my country. The traditional method for detecting goat pox can be agar diffusion method, but the sensitivity of this method is not high and the detection speed is slow.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects of low sensitivity and slow detection speed of the existing methods for detecting goat pox disease, and provide a diagnostic reagent for goat pox positive indirect hemagglutination and a preparation method thereof. , Rapid, accurate and simple detection of goat pox virus antibody.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

The goat pox virus antigen-sensitized red blood cell content in the goat pox positive indirect hemagglutination diagnostic reagent of the present invention is 0.6-1.2% (volume), and the purified goat pox virus antigen content is 20-200 μg/ml.

Preferably, the content of goat pox virus antigen-sensitized erythrocytes in the above-mentioned hemagglutination diagnostic reagent is 0.8-1%, and the content of purified goat pox virus antigen is 100 μg/ml.

The sensitized erythrocytes in the goat pox positive indirect hemagglutination diagnostic reagent are the alkylated sheep erythrocytes sensitized with purified goat pox virus antigen.

The present invention also provides a method for preparing the above-mentioned goat pox forward indirect hemagglutination diagnostic reagent: collect healthy sheep venous blood, prepare red blood cell suspension, carry out hydroformylation with glutaraldehyde, sensitize with purified goat pox virus antigen, After centrifugation, the precipitated erythrocytes are taken to prepare erythrocyte suspension, which is obtained after passing the hemagglutination test.

Further, the above method is as follows: collect venous blood from healthy sheep, extract erythrocytes by centrifugation with PBS, prepare erythrocyte suspension with phosphate buffer, formaldehydeylate with glutaraldehyde, and centrifuge to obtain erythrocytes, and use purified goat pox virus Antigen sensitization, centrifugal separation, the precipitated red blood cells are washed with phosphate buffer containing NaN ₃ and newborn calf serum, and then the buffer is used to prepare red blood cell suspension, which is obtained after passing the hemagglutination test.

Optimally, the aforementioned preparation method is as follows: collect venous blood from healthy sheep, extract red blood cells by centrifugation with PBS, then prepare a 2.5% (volume) red blood cell suspension with 0.1M, pH7.4 phosphate buffer, and use glutaraldehyde Centrifuge after hydroformylation, wash with 0.1M, pH 7.4 phosphate buffer to obtain formaldehyde erythrocytes, dissolve 10 times the volume of 0.1M, pH 7.4 phosphate buffer, and then mix with an equal volume Quantitatively purified goat pox virus antigens were mixed, stirred at room temperature for 2 hours for sensitization, centrifuged, and the precipitated red blood cells were washed with 0.1M, pH7.4 phosphoric acid solution containing 0.1% (mass) NaN ₃ and 1% (volume) newborn calf serum. After washing with the buffer solution, the red blood cell suspension is prepared with the buffer solution, and obtained after passing the hemagglutination test.

The method for detecting goat pox with the aforementioned diagnostic reagents: during detection, the pH value is controlled at 7.4-7.8, the reaction temperature is 20-25° C., and the reaction time is 50 minutes.

Compared with the existing commonly used agar diffusion test, the present invention prepares goat pox positive indirect hemagglutination diagnostic reagent by purifying goat pox virus antigen sensitized and aldehylated sheep erythrocytes, which can detect goat pox virus antibody quickly, accurately and easily. The results can be judged within 50 minutes, and antibodies with lower titers can be detected. It has good specificity, sensitivity and repeatability, and can be widely used in the rapid and large-scale detection of goat pox virus antibodies in veterinary clinics. , to provide a basis for disease prevention, diagnosis and control.

Detailed ways

Goat pox reverse indirect hemagglutination diagnostic reagent is prepared according to the following ratio and method

1. Preparation of aldylated red blood cells

Aseptically collect venous blood from healthy sheep, add 10 times the volume of PBS, centrifuge at 2000 rpm for 15 minutes, wash 5 times with PBS, and prepare 2.5% (volume) red blood cell suspension with phosphate buffer (0.1M, pH 7.4) , and then mix this erythrocyte suspension with 1% (volume) glutaraldehyde aqueous solution at a volume ratio of 9:1, stir slowly at room temperature for 1 hour, centrifuge at 2000 rpm for 15 minutes, and wash 5 times with phosphate buffer solution to obtain Aldehydization precipitates red blood cells.

2. Sensitization: Dissolve the formaldehyde-precipitated erythrocytes in 10 times the volume of phosphate buffer (0.1M, pH 7.4), mix with an equal volume of purified goat pox virus antigen, stir at room temperature for 2 hours, and centrifuge at 2000 rpm After 15 minutes, the precipitated sensitized red blood cells were obtained.

3. Wash the precipitated sensitized red blood cells with phosphate buffer (0.1M, pH 7.4) containing 0.1% (weight) NaN3 and 1% (volume) newborn calf serum for 5 times, and then make the concentration of sensitized red blood cells 1% ( volume), red blood cell suspension with viral antigen content of 100 μg/ml.

4. According to the conventional reverse indirect hemagglutination test method, the reverse indirect hemagglutination diagnostic reagent is prepared after passing the analysis and testing.

During detection, the pH value is controlled at 7.4-7.8, the reaction temperature is 20-25° C., and the reaction time is 50 minutes, which are the optimal reaction conditions.

Claims (7)

1. A diagnostic reagent for goat pox positive indirect hemagglutination, characterized in that: the content of goat pox virus antigen-sensitized erythrocytes in the hemagglutination diagnostic reagent is 0.6-1.2% (volume), and the content of purified goat pox virus antigen is 20-20%. 200 μg/ml. 2. According to the goat pox positive indirect hemagglutination diagnostic reagent of claim 1, it is characterized in that: the content of goat pox virus antigen sensitized red blood cells in the hemagglutination diagnostic reagent is 0.8～1%, and the content of purified goat pox virus antigen is 100 μ g /ml. 3. The goat pox positive indirect hemagglutination diagnostic reagent according to claim 1 or 2, characterized in that: the sensitized red blood cells are alkylated sheep red blood cells sensitized with purified goat pox virus antigen. 4. according to the preparation method of the goat pox forward indirect hemagglutination diagnostic reagent described in any one of claims 1 to 3, it is characterized in that: collect healthy sheep venous blood, be prepared into erythrocyte suspension, carry out hydroformylation with glutaraldehyde, It is sensitized with purified goat pox virus antigen, centrifuged, and precipitated red blood cells are taken to prepare red blood cell suspension, which is obtained after passing the hemagglutination test. 5. according to the preparation method of the goat pox forward indirect hemagglutination diagnostic reagent described in claim 4, it is characterized in that: collect healthy sheep venous blood, extract erythrocyte with PBS centrifugation, then prepare erythrocyte suspension with phosphate buffer, use Glutaraldehyde is hydroformylated and centrifuged to obtain aldolized erythrocytes, sensitized with purified goat pox virus antigen, centrifuged, and the precipitated erythrocytes are washed with phosphate buffer containing NaN ₃ and newborn calf serum, and then formulated with this buffer The red blood cell suspension is obtained after passing the hemagglutination test. 6. according to the preparation method of the goat pox positive indirect hemagglutination diagnostic reagent of claim 5, it is characterized in that: collect healthy sheep venous blood, extract red blood cell with PBS centrifugation, then use the phosphate buffer of 0.1M, pH7.4 Prepare 2.5% (volume) erythrocyte suspension, formaldehydeize with glutaraldehyde and then centrifuge, wash with 0.1M, pH 7.4 phosphate buffer to obtain formaldehyde erythrocytes, dissolve formaldehyde erythrocytes in 10 volume times 0.1M, pH 7.4 phosphate buffer, mixed with an equal volume of purified goat pox virus antigen, stirred at room temperature for 2 hours for sensitization, centrifuged, and the precipitated red blood cells were washed with 0.1% (mass) NaN ₃ and 1 % (volume) Newborn calf serum was washed with 0.1M, pH 7.4 phosphate buffer, and then this buffer was used to prepare red blood cell suspension, which was obtained after passing the hemagglutination test. 7. The method for detecting goat pox with a diagnostic reagent according to any one of claims 1 to 6, characterized in that: during detection, the pH value is controlled at 7.4-7.8, the reaction temperature is 20-25° C., and the reaction time is 50 minutes.